Keywords

10.1 Introduction

Talaromyces flavus is the most common species of the genus Talaromyces, which has been studied and applied as a biocontrol agent, a producer of secondary metabolites or enzymes. T. flavus is an extremely variable species found in soils and on organic materials that undergo slow decomposition. The species is widespread in its distribution but it is most commonly reported from the warmer regions of the world. It also occurs in foods, probably as a contaminant (Pitt and Hocking 2009). Dangeard (1907) provided a detailed report of Penicillium vermiculatum, which Benjamin (1955) relocated to the genus Talaromyces, and Orr et al. (1963) demonstrated the identity of this strain with Gymnoascus flavus described by Klocker (1902). Arachniotus indicus (Chattopadhyay and Das Gupta 1959) and P. liani were also declared identical with T. vermiculatus (Ghosh et al. 1961). Stolk and Samson (1972) redefined the genus Talaromyces and distinguished two new varieties of T. flavus, namely T. flavus var. flavus (isolates varies in color and colony characteristics; Fig. 10.1) and T. flavus var. macrosporus.

Fig. 10.1
A three-part image depicts the isolated Talaromyces flavus in three different colors of pale yellow, pale brown, and bright yellow.

Talaromyces flavus isolates showing different colony characteristics: (a) Tf1, (b) Tf2, and (c) Tf3

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10.2 Classification

Species classified in the phylum Ascomycota, class Eurotiomycetes, order Eurotiales, family Trichocomaceae, and genus Talaromyces are as follows (MycoBank 2010):

  • Talaromyces flavus (Klocker) Stolk and Samson (1972)

  • Basionym: Gymnoascus flavus Klocker (1902)

  • Synonyms: Arachniotus indicus Chattopadhyay and Das Gupta (1959)

  • Talaromyces flavus var. flavus (Klocker) Stolk and Samson (1972)

  • Synonyms: Penicillium liani Kamyschko (1962), T. vermiculatus (Dangeard) Benjamin (1955), G. flavus Klocker (basionym), A. indicus Chattopadhyay and Das Gupta (1959), A. indicus var. major Chattopadhyay and Das Gupta (1959)

Status conidialis (anamorphs):

  • P. vermiculatum Dangeard (1907)

  • P. liani Kamyschko (1962), Penicillium vermiculatum Dangeard (1907)

  • Synonym: Eupenicillium vermiculatum (Dang.) Ram and Ram (1972)

Teleomorphs:

  • P. dangeardii Pitt (1979)

  • T. flavus var. flavus (Klocker) Stolk and Samson (1972)

10.3 Secondary Metabolites

The organic soluble metabolites of this fungus include D-glucono-1,4-lacton, 5-hydroxymethylfurfural, 4,6-dihydroxy-5-methylphthalimide, methyl-4-carboxy-5-hydroxyphethalal dehydrate, hexaketide, 7-hydroxy-2,5-dimethylchromone, 3-hydroxymethyl-6,8-dimethoxycoumarin, altenusin, desmethyldehydroaltenusin, talaroflavone, deoxytalaroflavone, 2-methylsorbic acid, sorbic acid, bromomethylsorbic acid, and bromosorbic acid (Ayer and Racok 1990a). Some of the above-mentioned metabolites (2-methylsorbic acid, sorbic acid, bromomethylsorbic acid, and bromosorbic acid) play a fundamental role in the biogeochemical cycling of phosphorus (P) in natural and agricultural ecosystems. Some of the metabolites produced by the T. flavus are reported in Table 10.1.

Table 10.1 List of important metabolites produced by T. flavus
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10.4 Enzymes

Esterase/amidase from P. vermiculatum stereoselectively hydrolyzed only 2R,3S enantiomer from the synthetically prepared monoalkylesters or dialkylamides of racemic phosphomycin (Demain et al. 1972). Amine oxidase (EC 1.4.3.4) catalyzes the oxidative deamination of amines by the formation of aldehyde, hydrogen peroxide, and ammonia. T. flavus var. flavus isolated from a soil sample collected from a rice field produced amine oxidase stable up to 40 °C with the optimum pH in a range of 7.5–8.5. This enzyme was intended to be used in biochemical analysis, e.g., determination of the freshness of meat by assaying tyramine or other amines (Matsumoto and Takada 1984). α-Amylase and glucoamylase P. vermiculatum precultured on amylose gel and cultured on a medium containing corn starch secreted crude α-amylase (EC 3.2.1.1), which was further purified on a cross-linked starch (Augustín et al. 1983). Thermostable amylases are used for starch hydrolysis in the liquefaction step of industrial starch syrup production. T. flavus secreted a thermophilic glucoamylase (EC 3.2.1.3), which exhibited peak activity at 50 °C and a pH of 4.0–4.8 (Hang and Woodams 1993). Chitinase: T. flavus grown in the presence of chitin produced two chitinases (EC 3.2.1.14). The isolated enzymes with a molecular mass of 41 and 32 kDa decomposed the cell wall of Verticillium dahliae, Sclerotinia sclerotiorum, and Rhizoctonia solani (Duo-Chuan et al. 2005) at the optimum pH between 4.0 and 5.0, respectively and 40 °C. P. vermiculatum produced dextranase (EC 3.2.1.11) at the optimum temperature between 50 and 55 °C and pH in a range of 5.0–5.5. It hydrolyzed dextran to isomaltose, isomaltotriose, and glucose (Sun et al. 1988). The main application field of dextranases is sugarcane processing. The use of dextranases has also been extended to dental care as a toothpaste additive, since dextran has been shown to be involved in dental plaque formation (Galvez-Mariscal and Lopez-Munguia 1991). T. flavus var. flavus cultured aerobically at 28 °C for 48 h on a medium containing fructan-afforded fructanase (EC 3.2.1.80), which has been applied in the production of fructose from Jerusalem artichokes (Ishibashi et al. 1974). T. flavus secreted α-D-galactosidase in the presence of 6-deoxyglucose. The crude enzyme was composed of three isoenzymes. The most important isoenzyme, αGal-1, showed a different regioselectivity than the other two isoenzymes. Purified αGal-1 catalyzed the transglycosylation of tert-butanol and split off D-galactose from raffinose and stachyose. This enzyme, inhibited by α-D-galactopyranosylazide, D-xylose, melibiose, or lactose (Simerská et al. 2007), catalyzed the reaction of 4-nitrophenyl-α-D-galactopyranoside (LXX) and its 6-acetyl derivative LXXI to 4-nitrophenyl-α-Dgalactopyranosyl-(1→3)-6-O-acetyl-α-D-galactopyranoside (LXXII) (Simerská et al. 2003). A mixture of biosides, which composed of 86.5, 3.5, and 8.0% of LXXIII, α(1→2), and α(1→6) regioisomers, respectively, was prepared after incubation of LXX with α-D-galactosidase from T. flavus (Weignerová et al. 2001). P. vermiculatum, grown in a culture enriched with α-1,3-glucan, secreted a remarkable amount of α-1,3-glucanase (EC 3.2.1.84). The production of this enzyme was stimulated by the addition of surfactants Tween 80 or Tergitol NPX to the culture medium (Reese et al. 1972). The antifungal activity of this enzyme was studied in relation to its role in mycoparasitic processes, especially in degradation of the host cell wall (Sanz et al. 2004). Glucose oxidase (EC 1.1.3.4) catalyzes the oxidation of β-D-glucose to gluconic acid and hydrogen peroxide using molecular oxygen as the electron acceptor. T. flavus produces glucose oxidase, which is involved in the biocontrol of fungal plant pathogens. Pectinolytic enzymes are important for several industrial applications such as improving juice yields and clarity. Other areas of application include the paper and pulp industry, waste management, animal feed preparation, or the textile industry. T. flavus, precultured for 24 h on a solid substrate culture like passion fruit peel and then transferred into a new medium supplemented with 0.5–0.8% citrus pectin, secreted pectinesterase (EC 3.1.1.11) and polygalacturonase (EC 3.2.1.15) into the medium. High levels of pectinases cultivated in solid state fermentation using citrus pulp pellets were produced by T. flavus isolated from Brazilian soil (Siessere and Said 1989). Phytase, an enzyme that breaks down the indigestible phytic acid/phytates (inositol penta-, tetra-, and triphosphate) found in grains and oil seeds, was produced by T. flavus cultured in a medium containing up to 6% of saccharides and 2% of nitrogen-containing compounds (Jiang et al. 2007). Proteinase K (EC 3.4.21.64), a serine protease, is used in molecular biology because it rapidly inactivates nuclease, which might otherwise degrade the DNA or RNA during purification and maintains its activity in the presence of chemicals that denature proteins. Proteinase K was secreted by T. flavus in media containing goat hairs. The highest enzyme production was observed at pH 6.5 after a 9-day incubation and the isolated raw enzyme was separated into two fractions characterized by a molecular mass of 31.5 and 36.75 kDa, respectively (Mohawed and Badran 1995). The purified product was tested as an antifungal agent against brown spot disease caused by Botrytis fabae on a bean of Vicia faba (Haggag et al. 2006). α-L-Rhamnosidase T. flavus produced an extracellular α-L-rhamnosidase when incubated with inducers such as L-rhamnose, rutin, or naringin but not hesperidin (Monti et al. 2004). Fungal α-L-rhamnosidases have applications mainly in the food industry (Yadav et al. 2010). The enhancement of wine aroma by enzymatic hydrolysis of terpenylglycosides was also studied (Spagna et al. 2000). Cell wall-degrading enzymes, such as β-1,3-, β-1,4-, and β-1,6-glucanases, cellulase, and chitinase, are involved in the antagonistic activity of biocontrol agents against phytopathogenic fungi (Madi et al. 1997; Inglis and Kawchuk 2002). Some of the enzymes produced by Talaromyces flavus are listed in Table 10.2.

Table 10.2 List of the important enzymes produced by Talaromyces flavus
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10.5 Talaromyces flavus in Biological Pest Control

T. flavus suppresses Verticillium wilt of tomato (Dutta 1981), aubergine (Fahima and Henis 1995; Marois et al. 1982), potato (Fravel et al. 1986), or cotton (Nakova 2003) and parasitizes Sclerotium rolfsii (Madi et al. 1992, 1997), Sclerotinia sclerotiorum (McLaren et al. 1986), Rhizoctonia solani (Boosalis 1956), and Gaeumannomyces graminis var. tritici (Mohammadi and Ghanbari 2015).

Dethoup et al. (2007) characterized 122 isolates of Talaromyces flavus from 45 soil samples in 38 provinces of Thailand. Talaromyces flavus isolates were found in both non-agricultural and agricultural soil in Chiang Mai and Mae Hong Son provinces following heat and alcohol treatments. Twenty isolates of T. flavus were selected for antagonistic tests against 15 species of plant pathogenic fungi in vitro and in the greenhouse. All the selected isolates of T. flavus inhibited the mycelial growth of Phytophthora palmivora, P. parasitica, Peronophythora litchii, Colletotrichum capsici, C. gloeosporioides, Pestalotiopsis guepinii, Phyllosticta sp., Curvularia lunata, Helminthosporium maydis, H. oryzae, and Fusarium oxysporum. However, none of the isolates controlled Pythium aphanidermatum, Lasiodiplodia theobromae, Rhizoctonia solani, and Sclerotium rolfsii in vitro. However, in the greenhouse experiment, 20 isolates of T. flavus controlled stem rot of mung bean, caused by S. rolfsii, 7 and 14 days inoculation, and 6 isolates gave control up to 30 days inoculation. Bashyal (2018) reported that T. flavus is effective against Fusarium fujikuroi responsible for bakanae disease in rice (Fig. 10.2).

Fig. 10.2
A three-part image of the dual culture assay of Fusarium fujikuroi in different colors.

Dual culture assay of Fusarium fujikuroi with different isolates of Talaromyces flavus: (a) control, (b) Fusarium fujikuroi + Tf1, and (c) Fusarium fujikuroi + Tf2

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Cell wall-degrading enzymes, such as β-1,3-, β-1,4-, and β-1,6-glucanases, cellulase, and chitinase, are involved in the antagonistic activity of biocontrol agents against phytopathogenic fungi (Madi et al. 1997; Inglis and Kawchuk 2002). In addition, T. flavus antagonizes Verticillium dahliae by parasitism and antibiosis (Fahima et al. 1992; Marois et al. 1984). Microsclerotia of V. dahliae were suppressed by a culture filtrate of T. flavus and this effect was attributed to the action of glucose oxidase (Fravel and Roberts 1991; Kim et al. 1988). In a recent study, the effect of seed treatments of Talaromyces flavus on sugar beet seedling damping-off disease under greenhouse conditions reported that the isolates reduced the percentage of damping-off disease from 40 to 7.5% compared to infected control (Naraghi et al. 2012). Furthermore, in another field study, a significant decrease in the incidence of sugar beet seedling damping-off disease was observed along with increase in yield in treatments containing antagonistic fungi T. flavus and T. harzianum compared to the control (Naraghi et al. 2014; Lamichhane et al. 2017). Further, improved growth was reported on cotton and potato when they were treated with biocontrol agent Talaromyces flavus (Naraghi et al. 2012). Bashyal et al. (2020) reported increased drought tolerance in T. flavus-treated rice seedlings.

Chattopadhyay and Das Gupta (1959) isolated T. flavus from paddy rhizosphere and reported phosphate-solubilizing activity of T. flavus under in vitro conditions that positively influenced the growth of the rice, Cicer arientinum, and Vigna radiata under greenhouse conditions. He further reported that T. flavus have the capacity to convert insoluble phosphorus to soluble form.

Haggag et al. (2006) purified proteases from the culture filtrate of T. harzianum and T. flavus and tested for their antifungal activity against brown spot disease caused by Botrytis fabae on faba bean. T. flavus exhibited high levels of extracellular protease activity compared with T. harzianum. Germination and growth rate, extracellular polygalacturonase (PGase), and carboxymethyl cellulase (CMCase) activities of Botrytis fabae were inhibited by the purified protease at a concentration of 40–120 U/ml. Growth and extracellular production of B. fabae were completely inhibited by the protease enzyme of T. flavus at a concentration of 80 U/ml, while protease from T. harzianum was effective at 120 U/ml. Proteases were effective in reducing brown spot disease severity and pathogen sporulation on faba bean leaves inoculated with B. fabae.

Glucose oxidase, secreted by T. flavus, retards hyphal growth and kills microsclerotia of V. dahliae in vitro, probably by generating toxic peroxide (Kim et al. 1988; Stosz et al. 1996), but only if a sufficient amount of glucose is available (Murray et al. 1997). Partial disintegration of melanin was observed near hyphae of T. flavus colonizing sclerotia of S. sclerotiorum (McLaren et al. 1989). The inhibition of germination and melanin formation in sublethally heated microsclerotia of V. dahliae and additive suppression by sublethal heating and T. flavus treatment was studied (Tjamos and Fravel 1995). A glucose oxidase gene was located and isolated, and mutants of T. flavus with both high and low production ability of this enzyme were constructed. A high level of glucose oxidase in tobacco and cotton as a result of the expression of its gene from T. flavus was associated with phytotoxic effects such as reduced root growth, slow germination on culture medium, or reduced lateral root formation (Murray et al. 1999).

To increase the effectiveness of T. flavus isolates obtained from greenhouse cucumbers and field-grown tomatoes, five chemical stabilizers were evaluated. Based on the results of previous studies, the most effective substrate for the growth, sporulation, and stability of T. flavus isolates related to the above-mentioned plants was a mixture of rice bran and peat moss. Different chemical stabilizers were mixed with the above-mentioned substrate containing spore suspensions of various T. flavus isolates. Completely randomized experiment was conducted under greenhouse conditions with seven treatments and three replications. The results of this study indicated that treatments containing sodium nitrate and D-cycloserine were more effective than those containing other stabilizers (Bahramiyam et al. 2016).

Fravel et al. (1986) evaluated the use of pyrophyllite clay (Pyrax), milled chitin, maize cobs, fish meal, neem cake, groundnut hulls, soya fiber, and wheat bran to make alginate prill with or without ascospores of T. flavus. The formulations were compared for their ability to induce T. flavus to control Verticillium wilt of aubergine in the greenhouse in field soil and to increase populations of T. flavus in three field soils (two loamy sands, one silty clay). Survival of T. flavus in prill at 5 °C or ambient temperature (22–24 °C), as well as the carbon (C) and nitrogen (N) contents of the prill, was also determined. Two formulations (maize cobs and pyrophyllite) consistently enhanced biocontrol activity.

10.6 Conclusion

Talaromyces flavus is a significant soil-inhabiting fungus that produces important metabolites and enzymes that have established their effects on biological models. The application of its enzymes has enabled stereoselective preparation of many useful saccharides. T. flavus is a very variable species and not all of its secrets have as yet been uncovered. At the same time, this microbe was utilized as biological control agent against many important diseases. However, genomic studies are lacking on this fungal species. Further, emphasis should be given on genes involved in secondary metabolite production, whole genome studies of this microbe, and tripartite interaction to understand this fungal species further.