Applied Mycology

Adrian R Radillo

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This book is intended to provide both students and researchers with a broad background to some of the fastest developing areas in current applied mycology. It was clearly not going to be practical to incorporate all of the different aspects and directions of such a broad area into a single volume, and so we have brought together a range of contributions to highlight the diverse nature of current applied mycology research. Environmentally, fungi are of vital importance and their activities are closely linked with those of bacteria and lower plants in undertaking the nutrient and chemical cycles needed to maintain life on Earth. Despite such fundamental importance, mycology is frequently overlooked in many scientific courses and treatments.

Figures (57)

Fig. 2.1. Seasonal changes in the relative spore density of Glomus fasciculatum and G. intraradices (F+l), Acaulospora spp. (A), Glomus spp. (G) and other spores (Ot) at the four sites studied. Bars represent the standard error of the mean (from Escudero and Mendoza 2005).

Table 2.1. Effect of soil waterlogging and drought on AM fungi variables and nodules in Lotus tenuis roots, and spore and hyph soil compared with field capacity (from Garcia et al., 2008).

Table 2.2. Effect of drought stress and microbial treatment on mycorrhizal and rhizobial symbiosis development (from Ruiz-Lozano et a/., 2001). SRihneneeretin: Betares ee ems Seapets Te AAA, NRG a Nem shencominrsae ee The effect of drought stress and microbial treatment on mycorrhizal and rhizobial symbiosis development was reviewed by Ruiz-Lozano et al. (2001), and they suggested that an alleviation of oxidative damage is strongly involved in AM protection against nodule senescence. In dual colonization experiments the colonization frequency (F) in soybean roots was similar during all the mycorrhizal treatments used, and this was not affected by the presence or absence of Bradyrhizobium japonicum or by drought stress (see Table 2.2).

Fig. 2.4. Shoot N (a) and P (b) contents (mg/plant) in non-mycorrhizal (control) and mycorrhizal (Glomus sp. and G. deserticola) lettuce plants grown at three levels of salinity (1.1, 1.4 and 1.7 dS/m) (from Ruiz-Lozano and Azc6n, 2000). antagonistic effect of Na on chlorophyll synthesis (Giri and Mukerji, 2004). The detrimental effect of Na is a result of its ability to compete with K for binding sites essential for various cellular functions. Potassium activates a range of enzymes, and Na cannot substitute in this role. Thus, high levels of Na or high Na/K ratios can disrupt various enzymatic processes in the cytoplasm. Mycorrhizal Acacia and Sesbania plants have been found to have a higher concentration of K in root and shoot tissues at all salinity levels (Giri and Mukerji, 2004; Giri et al., 2007). Similar results were reported by Sannazzaro et al. (2006) in L. tenuis plants colonized by G. intraradices. It seems that higher K accumulation by AM plants under salt-stress conditions

Table 3.2. Legal limits for ochratoxin A in different food products as set by the European Commission (Commission Regulation (EC) No.123/2005 of 26 January 2005). Following evaluations of OTA carried out in the 1990s, the Joint FAO/ WHO Expert Committee on Food Additives (JECFA) has established a provisional tolerable weekly intake of 100 ng/kg body weight (b.w.) per week for OTA (JECFA, 2001). The World Health Organisation (WHO) has proposed a provisional tolerable daily intake of 16 ng/kg b.w., and this was lowered to 5 ng/kg by the EC Scientific Committee on Food in 1998 in view of the carcinogenicity of OTA. More recently, the Panel on Contaminants in the Food Chain of the European Food Safety Authority (EFSA) concluded that, according to the estimated exposure levels (2-8 ng/kg b.w./day), exposure to OTA varies between 15 and 60 ng/kg b.w./week. Based on the evaluation of the toxicity of OTA, the EFSA established a tolerable weekly intake of 120 ng/ kg b.w. for OTA (EFSA, 2006). The main contributors to OTA intake in the EU are cereals and cereal products and, to a lesser degree, wine (see Chapter 9, this volume), coffee, beer, pork, pulses and spices (EFSA, 2006).

Table 3.3. Ochratoxin-producing Aspergillus species.

Fig. 3.2. A proposed pathway of ochratoxin biosynthesis (adapted from Huff and Hamilton, 1979; Harris and Mantle, 2001; O’Callaghan and Dobson, 2005). SAM, S-adenosyl methionine; PKS, polyketide synthase; P450, cytochrome P450-containing enzyme; CPO, chloroperoxidase.

Fig. 3.3. Degradation of ochratoxin A by carboxypeptidase A.

Fig. 4.3. Dose effect of Pseudomonas fluorescens on the percentage of Douglas fir short roots mycorrhiza with Laccaria bicolor S238, 23 weeks following fungal and bacterial inoculations. Fungal inoculation dose, 50 mg dry weight mycelium/m?; ™ fungal inoculation dose, 100 mg dry weight mycelium/m?. For each fungal inoculation dose, columns indexed by the same letters are not significantly different according to a Scheffe test (from Frey-Klett et a/., 1999).

'Data in the same line followed by the same letter are not significantly different, according to Student's t-test (—p < 0.05). Table 4.2. Biological and chemical characteristics of powders from termite mounds (from Duponnois et al., 2005).

'Data in the same line followed by the same letter are not significantly different, according to the Student's t-test (p < 0.05).

Table 4.6. Effect of ectomycorrhizal inoculation and termite mound powder amendment rates on Acacia holosericea growth and on mycorrhizal formation after 2 months’ culture in 1 | pots (from Duponnois, 2007).

Table 5.1. Water-borne conidial fungi recovered from tree canopy habitats.

References: 1, Ando and Kawamoto, 1986; 2, Ando and Tubaki, 1984a; 3, Ando and Tubaki, 1984b; 4, Bandoni, 1981; 5, Czeczuga and Ortowska, 1994; 6, Czeczuga and Ortowska, 1998a; 7, Czeczuga and Ortowska, 1998b; 8, Czeczuga and Ortowska, 1999; 9, Génczél, 1976; 10, G6énczdl and Révay, 2003; 11, Génczdl and Révay, 2004; 12, Génczdl and Révay, 2006; 13, Karamchand and Sridhar, 2008; 14, Kaufman et al., 2008; 15, Mackinnon, 1982; 16, Magyar et a/., 2005; 17, Sokolski et a/., 2006; 18, Sridhar et a/., 2006. and Petrini, 1984), in medicinal plants (Raviraja et al., 2006), in tropical fores trees (Suryanarayanan et al., 2003; Murali et al., 2006) and in mangrove plant and mangrove associates (Ananda and Sridhar, 2002; Maria and Sridhai 2003). Several endophytic fungal associations with plants are antagonistic t insect herbivores, and the canopy endophytes may protect plant specie from herbivores (Carroll, 1991; Azevedo et al., 2000). Many canopy fung produce secondary metabolites and the bark endophyte, Taxomyces andreanae has been considered for in vitro production of the anticancer agent taxo (Stierle et al., 1993; Strobel et al., 1993). NAAO wt nAR et aT TTOOR\ hace Anncancaitoale nckivesatad fhatk awe de TO!

Table 6.1. Species number, relative proportion in decomposer fungal assemblages and genera of endophytic phyllosphere fungi occurring on leaf litter of tree species. 4 Species number of endophytic fungi that occurred frequently on leaf litter as compared with the total species number of endophytic fungi on liv > Relative proportion of endophytic fungi in decomposer fungal assemblages in terms of frequency, given as: (the sum of isolation frequency of ¢ fungi)/(the sum of isolation frequency of all fungi on leaf litter) x 100. © Genera of endophytic fungi that occur on leaf litter. St, Stachylidium; Sp, Sphaeriaceous; Al, Alternaria; Ni, Nigrospora; Pe, Pestalotiopsis; Ds, ¢ mycelium; Ma, Macrophoma; Co, Coniothyrium, Cl, Cladosporium; Cr, Cryptocline; Gl, Gloeosporidiella, Le, Leptostroma; Ph, Phomopsis; Cm, Colletotrichum; Gu, Guignardia; Di, Discostroma; Ge, Geniculsporium; Xy, Xylaria, As, Ascochyta; Rh, Rhytismataceous. 4 References : 1, Ruscoe (1971) ; 2, Watson et al. (1974); 3, Wildman and Parkinson (1979); 4, Cabral (1985); 5, Heredia (1993); 6, Sieber-Cans Sieber (1993); 7, Okane et al. (1998); 8, Osono (2002); 9, Osono et al. (2004); 10, Koide ef al. (2005b); 11, Osono et al. (2009).

Fig. 6.1. (a) A bleached leaf litter of Camellia japonica. Filled arrows, colonies of Coccomyce: sp.; open arrows, colonies of Lophodermium sp. Bar = 1 cm. (b) Neighbour-joining tree based on sequences of the rDNA-ITS region from Coccomyces spp. associated with seven different host tree species in a subtropical forest in southern Japan. Numbers above the branches are bootstrap values. Lophodermium pinastri sequence was used as outgroup.

Fig. 7.1. The cell cycle of Saccharomyces cerevisiae. and M (see Fig. 7.1; Wheals, 1987). G, and G, represent two ‘gap’ periods of variable length, during which organelle production and normal cell processes such as growth and development take place. G, represents the time prior to starting anew round of cell division. At the end of G, a decision point, termed START, represents a collection of cellular events necessary for division. One important issue before the start of the cell cycle is whether the environmental conditions are positive for replication. Also, a minimal size has to be reached. DNA synthesis occurs during the S phase. The post-synthetic G, phase coincides with

Fig. 7.2. A schematic overview of the lectin-binding model of flocculation and the factors affecting flocculation according to their mode of operation (from Verstrepen et al., 2003b).

Fig. 7.3. Interrelation between yeast metabolism and the production of flavour compounds.

Table 7.2. Important flavour compounds produced by yeasts, their concentration range in lager beer and their flavour threshold (from Engan, 1972; Meilgaard, 1975).

Fig. 7.4. Schematic presentation of the formation, reassimilation and reduction of diacetyl.

Fig 7.5. The continuous fermentation plant of Dominion Breweries. MVs, maturation vessels. ‘facilitated cell recovery and re-use, and simplified downstream processir Verbelen et al., 2006). Different immobilization types can be used, includir nclusion in hydrogels, adsorption on porous and non-porous carriers, sel aggregation (flocculation) and containment behind membranes. In continuot mmobilized yeast fermentation systems, different types of bioreactors a 1ow being used, such as packed-bed reactors, fluidized bed reactors, airl reactors and stirred reactors. The choice of bioreactor is related to the type. mmobilization, to the metabolism of cells and to the mass and heat transf requirements (Obradovic et al., 2004). At present, only beer maturation and alcohol-free beer production a a er) 1° #Y 1

Unlike furfural, knowledge of HMF conversion has been limited because there has not been a readily available commercial source for an HMF conversion product. Following the furfural conversion route, it has been assumed that HMF is converted to HMF alcohol (Nemirovskii et al., 1989). Recently, an HMF metabolic conversion product was isolated and identified

Fig. 8.3. The genomic adaptation to HMF stress. Interactions of significantly expressed genes of ethanologenic yeast Saccharomyces cerevisiae under a normal control condition and 5-hydroxymethylfurfural (HMF) stress condition from 0, 10, 30, 60 and 120 min after the treatment, showing significantly induced (blue) and repressed (red) mRNA expression causec by the HMF stress on a defined medium. Yellow colour indicates mRNA equally expressed under different conditions. Varied colours between yellow and red, or yellow and blue, indicate varied relative quantitative measurements of MRNA expression levels in a log scale.

Fig. 8.4. Computation modelling of gene regulatory networks of HMF stress. Temporal interactions for a subset of 46 genes in response to HMF for biomass conversion to ethanol by the ethanologenic yeast. The p-values of each edge are displayed. A solid directed edge in green from the first gene node to the second gene node with an arrowhead indicates enhancement of the second gene by the first gene; an edge in red from the first gene node to the second gene node with a solid dot indicates repression of the second gene by the first gene. The dashed edges represent the external influence from HMF to each gene: red for repressing and green for enhancing (from Song and Liu, 2007). Three known transcription factors, PDR1, PDR3, and YAP1, were examined and, in this subset, YAP1 was shown by the model to be one of the most influential regulators in the early response to HMF stress (see Fig. 8.4). This is strongly supported by current knowledge and documented experimental observations (Teixeira et al., 2006). For example, the following edges have been reported as transcriptional regulations, including YAP1 to

Fig. 9.1. Chemical structure of ochratoxin A.

Fig. 9.2. Factors affecting grape colonization by OTA-producing fungi.

Fig. 10.1. Backbone structure of B-b-glucans from Ganoderma lucidum.

Fig. 10.2. Triterpenes identified from Ganoderma lucidum.

Table 10.4. Hepatoprotective effects of Ganoderma extracts and compounds. Table 10.4. Hepatoprotective effects of Ganoderma extracts and compounds. 2004). A hepatoprotective effect on ethanol-induced liver damage has also been reported for a hot-water extract of G. lucidum (Lee et al., 1998). It was suggested that this effect may be due to a superoxide scavenging activity, as animals treated with the extract had decreased levels of malonic dialdehyde (Lee et al., 1998). A G. lucidum extract (GLE) has been reported to improve CCL1,-induced liver fibrosis, leading to increases in plasma albumin and the albumin/globulin ratio, a reduction in the hepatic hydroxyproline and decreased activities of transaminases (Lin and Lin, 2006). In addition, GLE treatment may decrease expression of transforming growth factor-f1 (TGF-B1) and methionine adenosyltransferase (MAT1) (Lin and Lin, 2006). A polysaccharide isolated from G. lucidum has been reported to reduce the serum aspartate and alanine transaminases, alkaline phosphatase, total

Table 10.5. Cardioprotective effects of Ganoderma extracts and compounds. One of the possible mechanisms for reducing cholesterol is the inhibition of enzymes involved in cholesterol biosynthesis. Lanosterol analogues have been identified among G. lucidum triterpenes, and these compounds can act as dual inhibitors by suppressing lanosterol 14a-methyl demethylase (P-450DM), and as partial inhibitors of HMG-CoA reductase (Frye and Leonard, 1999). Ganoderic acid C, its derivatives and 26-oxygenosterols (ganoderol A and B, ganoderal A and ganoderic acid Y) can therefore potently inhibit lanosterol 14a-demethylase (Komoda et al., 1989; Hajjaj et al., 2005). As mentioned above, 7-oxo-ganoderic acid Z and 15-hydroxy-ganoderic acid S can suppress the activity of HMG-CoA reductase (Li et al., 2006). Ganoderic acid F has been reported to give some atherosclerosis protection due to the inhibition of an angiotensin-converting enzyme (Morigiwa et al., 1986), and ganodermic acid S has been shown to inhibit platelet aggregation (Su et al., 1999).

Table 10.6. Anti-diabetic effects of Ganoderma extracts and compounds. effects (van der Hem et al., 1995). Ganoderma lucidum polysaccharides (GLPS) have been reported to increase insulin and reduce glucose serum levels in a dose-dependent manner in alloxan-induced diabetic mice (Zhang et al., 2003) and, interestingly, GLPS was also found to inhibit alloxan-induced activation of NF-«B in the pancreas (Zhang et al., 2003). GLPS was able to decrease serum glucose and triglyceride levels in streptozotocin-induced diabetic mice, and improved the renal morphometric changes and oxidative stress state of diabetic mice. These observations suggest that GLPS may prevent or delay the progression of diabetic renal complications (He et al., 2006). A hypoglycaemic effect has also been shown for GLPS in normal mice, through its insulin-releasing activity. This was due to a facilitation of Ca** inflow to the pancreatic beta cells (Zhang and Lin, 2004). The commercially available G. ucidum water- soluble polysaccharides (Ganopoly) have been shown clinically to decrease the postprandial blood glucose levels in type-2 diabetic patients (Gao, Y. et al., 2004), although the major compounds responsible for the hypoglycaemic effects have been identified as peptidoglycans (Zhou et al., 2007).

lable 11.1. Clinical syndromes, associated dematiaceous fungi and suggested therapy. 2001). Specifically, in the yeasts Cryptococcus neoformans and Exophiala dermatitidis (syn. Wangiella dermatitidis), disruption of melanin production leads to markedly reduced virulence in animal models (Kwon-Chung eft al., 1982; Dixon et al., 1987). Melanin has also been shown to reduce the susceptibility of C. neoformans and H. capsulatum to amphotericin B and caspofungin, possibly by binding these drugs (van Duin et al., 2002; Ikeda et al., 2003). This effect is not apparent with azole drugs (van Duin et al., 2002). Finally, melanin has even been found in the yeast Candida albicans, suggesting that it may be a more fundamental and common virulence factor than previously thought (Morris-Jones et al., 2005).

Fig. 12.1. Antagonistic ability of 7; harzianum strains over-producing (P2.32) or lacking (R1 pH regulator PAC1. A, effect on enzymes and metabolites secreted by different strains. Wilc type (WT), P2.32 and R13 cultures were grown on PPG plates, buffered at pH 5.5 and previously covered with cellophane, which was removed after 3 days; 5 mm mycelial discs « Rhizoctonia solani strain or spores of Botrytis cinerea were placed on plates. Radial growth phytopathogen was measured at 48 h and then every 24 h for 5 days. Control: PPG plates where T. harzianum had not been previously grown. B, effect on mycoparasitism. Spores fre WT, P2.32 and R13 strains were inoculated on to MM with 0.2% glucose as the only carbor source and buffered at pH 5.5. Mycelial discs (5 mm) of FR. solani or B. cinerea were placed on the opposite side of Petri dishes. Control: plates without 7. harzianum (from Moreno- Mateos et al., 2007, with permission).

Fig. 12.2. Antimicrobial activity of aglycones in phenolic fraction from leaves of cucumber seedlings in which roots were either not colonized (light grey) or colonized by T-203 (dark grey) and then challenged with Pseudomonas syringae pv. lachrymans (Psl). Seedlings were of the same age and were extracted 2 days after challenge with Psl. These aglycones have antimicrobial activity against Psl, Agrobacterium tumefaciens (Agr), Bacillus megaterium (Bac), Micrococcus luteus (Mic), Saccharomyces cerevisiae (Sac), Fusarium oxysporum (Fus), Botrytis cinerea (Bot), Trichoderma asperellum (Trich) and Penicillium italicum (Pen) (from Harman et al., 2004a, with permission).

Fig. 12.3. Light micrography of ultra-thin cross-section of tomato root colonized by Trichoderma harzianum magnified x 2000. Arrows show hyphae stained by toluidine blue (from Chacén et al., 2007, with permission).

Fig. 12.4. Survival of tobacco plantlets after incubation for 1-4 weeks with Rhizoctonia solani. Wt, untransformed control; Bin, plants transformed with the empty vector; pT19.1 and pT20.1, transformants carrying the B-1,3-glucanase gene from T. harzianum. Figures bearing the same superscript letters have no significant differences (from Rincén, 2004, with permission). a © ie ee Recent findings on ISR stimulation i in plants have widened the range o characteristics that could be considered for improvement in BCAs. Fo: example, the selection of BCAs for plant root colonization would be enhancec by the isolation of strains that over-express ABC transporters, hydrophobin: and repellents. In T. harzianum CECT 2413, over-expression of the hydrophobin gene qid3 results in spores with improved adherence propertie: (Rosado et al., 2007). Finally, heterologous expression of genes such as thos« that encode for Trichoderma avr proteins may be useful for engineering plant: to increase disease resistance. Trichoderma gene expression may possibly become more effective at inducing plant resistance than the expression o

4722 saplings took up significantly more metal. > Control saplings took up significantly more metal (P = 0.05).

Fig. 13.2. General schematic representation of Agrobacterium-mediated transformation. Following activation and autophosphorylation of VirA, VirG is phosphorylated and induces vir gene transcription. A single-stranded T-DNA molecule is released after nicking by VirD1/VirD: border-specific endonucleases. The T-DNA border sequence also serves as the covalent attachment site for the VirD2 protein. Export of the virD2/T—strand complex to the host cell occurs via a type 4 secretion system (SS) formed by the VirB and VirD4 proteins. Several other Vir proteins (VirD5, VirE2, VirE3 and VirF) are independently transported into the host cell cytoplasm by the same VirB/VirD4 channel. Once inside the host cell, the virD2/T-strand conjugate is coated with VirE2 molecules, imported into the host cell nucleus and integrated into the host cell genome. NPC, nuclear pore complex.

Table 13.1. Commonly used binary vectors.

Table 13.2. Commonly used Agrobacterium tumefaciens strains. @Generally accepted classification scheme of Agrobacterium strains. Indicates opine catabolism of the original progenitor wild-type strain and/or non-disarmed parental Ti plasmid. Does not necessarily indicate opine production.

Fig. 13.3. Targeted gene disruption using two-marker selection. A second selection marker (2nd SM) is incorporated into the T-DNA gene-disruption cassette, illustrated at the left border, but it can be placed on either side of the disruption cassette. Following AMT, transformants are screened using both markers. If recombination occurred via the homologous recombination pathway, the second selection marker gene is lost. If the T-DNA insertion was via non- homologous recombination, the second selection marker gene is retained. (Sugui et al., 2005), Ceratocystis resinifera (Loppnau et al., 2004), Coccidioides posadasii (Kellner et al., 2005; Li et al., 2007), Fusarium graminearum (Malz et al., 2005), Fusarium oxysporum (Khang et al., 2005, 2006), Glarea lozoyensis (Zhang et al., 2003; Lu et al., 2005), Histoplasma capsulatum (Sullivan et al., 2002; Marion et al., 2006), Kluyveromyces lactis (Bundock et al., 1999), Leptosphaeria maculans (Gardiner and Howlett, 2004; Eckert et al., 2005; Gardiner et al., 2005; Blaise et al., 2007), Maganaporthe grisea (Khang et al., 2005, 2006), Mycosphaerella graminicola (Zwiers and De Waard, 2001), Ophiostoma piliferum (Hoffman and Breuil, 2004), Trichoderma atroviride (Cardoza et al., 2006), Verticillium dahliae (Chen and Fukuhara, 1988; Klimes et al., 2006) and V. fungicola (Amey et al., 2002). Gene-targeting efficiencies ranged from 14 to 90%, which are higher than those normally achieved by traditional transformation methods. Taroeted scene dienintion with AMT hac heen cambined with doiuble-

Table 14.1. Examples of metal nanoparticles produced from fungi. CdS, cadmium sulfide; ZrO,, zirconia; SiO,, silica; TiO,, titania; Ag, silver; CdSe, cadmium selenide; Au, gold; Pt, platinum.

Table 15.1. Chitinase genes isolated from fungi and yeasts. and 1. reesei, respectively (Jones ef al., 2004; Seidl et al., 2005). Chitinase genes of fungi can be functionally expressed in various hosts, such as E. coli, yeasts, filamentous fungi and plants. Expression of ech42- encoding endochitinase of T. harzianum in E. coli results in high chitinase activity (Carsolio et al., 1994). Transformation of Schizosaccharomyces pombe with cts1 from Saccharomyces cerevisiae results in the appearance of about a five- to 13-fold increase in chitinase activity (Kuranda and Robbins, 1987). Introduction of an Aphanocladium album chitinase gene into Fusarium oxysporum results in high chitinase levels in the host (Blaiseau et al., 1992). Expression of a gene-encoding endochitinase from T. harzianum in T.

able 16.1. Properties of extracellular proteases purified from mycoparasitic and nematophagous fungi.

Table 16.2. Characteristics of extracellular protease-encoding genes from mycoparasitic and nematophagous fungi.