Trichoderma

Biology and Applications

Prasun K Mukherjee, Uma Shankar Singh, Benjamin A Horwitz, Monika Schmoll, Mala Mukherjee, Prasun K Mukherjee, U S Singh, B A Horwitz, Monika Schmoll, Mala Mukherjee

• Éditeur: CAB International
• Année: 2013
• ISBN:
  9781789244663
• Sujet: Technologie et ingénierie
• Sous-sujet: Agronomie

Trichoderma spp. are biotechnologically significant fungi, being widely used both in agriculture and industry. These microbes are also a potential drug source of clinical importance. In recent years, driven by advances in genetics and genomics, research on these fungi have opened new avenues for its varied applications. Divided into three sections, covering taxonomy and physiology, interactions with plants and applications and significance, this book also discusses topics that have seen rapid developments in the recent years. Various aspects of Trichoderma like molecular taxonomy, sexual and asexual developments, secondary metabolism, beneficial interactions with plants, applications as cell factories and harmful interactions with humans are discussed. This book, thus, hopes to be an essential ready reference for researchers, students and people form industry as well.

1 Trichoderma in Agriculture, Industry and Medicine: An Overview

Prasun K. Mukherjee,¹* Benjamin A. Horwitz,² Uma S. Singh,³ Mala Mukherjee¹ and Monika Schmoll^4
1Central Institute for Cotton Research, Nagpur, India; ²The Technion – Israel Institute for Technology, Haifa, Israel; ³International Rice Research Institute, New Delhi, India; ⁴Austrian Institute of Technology GmbH (AIT), Tulln, Austria

1.1 Introduction

Species of the genus Trichoderma belong to one of the most useful groups of microbes to have had an impact on human welfare in recent times. These filamentous fungi have many applications (Fig. 1.1). They are the most widely used biofungicides and plant growth modifiers, and are sources of enzymes of industrial utility, including those used in the biofuels industry. Furthermore, they are prolific producers of secondary metabolites, some of which have clinical significance, and some species have been engineered to act as microbial cell factories for the heterologous production of important proteins. In the soil, Trichoderma species are used in the bioremediation of organic and inorganic wastes including heavy metals (Schuster and Schmoll, 2010; Harman, 2011a,b).

No doubt, the volume of literature (Google Scholar database ~138,000 hits) and the number of patents (Google patents database ~56,100 hits) being filed/granted on Trichoderma have seen exponential growth in the past few decades. Centuries ago, this genus was erected by Persoon (1794); however, the potential in human welfare was realized only in the 1930s after Weindling (1932, 1934) demonstrated that these fungi are mycoparasites, antibiotic producers and plant disease biocontrol agents. There was little research in the area of biological control using Trichoderma for the next three decades but then it picked up again, thanks to the pioneering and dedicated work of several research groups who demonstrated the bio-efficacy of Trichoderma under field conditions in the 1970s. Today, these fungi are among the most widely researched microbes, as is evident from the huge volume of published literature. Unfortunately, the taxonomy of these economically important fungi, which relied mostly on their morphology, was rather messy until the advent of molecular tools and barcodes (http://www.isth.info/). Even today, many of the strains deposited in culture collections across the world and many DNA sequences deposited in the databases are yet to be corrected (see Chapter 2, this volume). Correct identification (often neglected) is a must for these economically and clinically significant fungi. This is especially important for two reasons: (i) some of the beneficial traits are species (and often strain) specific and hence it is important to identify the species/strain to realize its full potential; and (ii), more importantly, it is necessary for the safety of the handler and the users (many are illiterate farmers/farm workers). Some species/strains are producers of mycotoxins, whereas others are human pathogens, especially threatening immuno-compromised individuals (see Chapter 17, this volume). It is unfortunate that even with the advent of freely available online software tools such as TrichoKey, TrichoBLAST (http://www.isth.info/) many of the widely used commercial strains are not yet correctly identified. This is one aspect that needs the immediate attention of scientists, government regulators and the production industry.

Fig. 1.1. Impact (positive and negative) of Trichoderma on human welfare.

1.2 Trichoderma in Agriculture

Trichoderma spp. have had a major impact on agriculture. Although their interaction with plants is not a strict symbiotic one as with rhizobia and mycorrhiza, yield improvement and control of soil-borne pathogens is striking. According to a conservative estimate, about 60% of all the registered biofungicides worldwide are Trichoderma based (Verma et al., 2007). In India alone, more than 250 Trichoderma- based formulations are sold commercially and many are added each year (Singh et al., 2012). This is in sharp contrast with other countries, mainly the industrialized nations, where only a handful of products are available. This can be explained by the fact that, in industrialized nations, Trichoderma are produced in large fermenters that need huge investment, whereas in India and perhaps in other developing nations, most of the companies produce Trichoderma on grains and, because the costs of labour are low, the set-up needs very small initial investments and the returns are high (Verma et al., 2007). Selling Trichoderma has thus become a very profitable business with small entrepreneurs. The adverse side effect of such advancements is the quality control, which is quite often compromised (use of Trichoderma as biofungicides is discussed in detail in Chapter 14, this volume). One interesting thing about Trichoderma is that it can control fungi belonging to taxonomically diverse groups as well as oomycetes. The plant defence induced by Trichoderma helps in restricting pathogenic bacterial growth on foliage (see Chapter 10, this volume; Harman et al., 2004; Druzhinina et al., 2011). Sustainable growth of the Trichoderma-based biofungicide market will require some modifications, however. One of the major issues is the availability of quality products, especially in developing countries. There is a need for improving the quality of formulations for higher initial colony-forming units (CFUs), extended shelf-life at ambient temperatures and efficacy under conditions of abiotic stresses (e.g. high soil pH, salinity and low moisture). In addition, there should be more exploratory research to identify new Trichoderma strains with novel applications. One example worth mentioning is the evaluation of marine isolates of Trichoderma for the biological control of plant diseases in saline and arid soils (Gal-Hemed et al., 2011). The isolation of endophytic Trichoderma strains is an emerging area that is yielding interesting results, including isolates that can confer biotic and abiotic stress tolerance and produce novel secondary metabolites (see Chapter 9, this volume; Bae et al., 2008, 2009, 2011).

Biocontrol is a complex mechanism mediated by direct parasitism (hyphae and resting structures/fruiting bodies) of plant pathogens, production of secondary metabolites, induction of plant defences, competition for iron and other nutrients, and consumption of germination stimulants from seeds/rhizosphere (Harman et al., 2004; Harman, 2006; Howell, 2006; Shoresh et al., 2010). Even though it is well researched, the role of individual mechanisms in vivo is not well understood. Gene function studies with individual gene knockout mutants have set the pace for understanding the mechanisms of biocontrol at the molecular level (for a review see Mukherjee et al., 2012a). Now that we have the genome sequences available for seven species of Trichoderma (with more species scheduled for sequencing already), it is expected that our understanding of biocontrol in a holistic manner will become more robust in the next few years (Druzhinina et al., 2011; Kubicek et al., 2011).

Apart from biocontrol, Trichoderma spp. are long known to improve plant (root and shoot) growth (Harman et al., 2004; Shoresh et al., 2010), recently shown to be through the production of phytohormones and certain other secondary metabolites (see Chapter 10, this volume; Lorito et al., 2010). It has also been proposed that Trichoderma improves plant growth through nutrient solubilization and hence shows better effects under nutrient stress conditions (Mastouri et al., 2010, 2012; Shoresh et al., 2010). These fungi colonize plant roots internally and externally and take up sucrose from roots, but simultaneously enhance photosynthetic ability and induced defence (Vargas et al., 2009, 2011). The plants also respond by inducing many stress-responsive genes (Alfano et al., 2007; Brotman et al., 2012; Moran-Diez et al., 2012). In addition to imparting tolerance to several abiotic stresses such as drought, salinity and so on, Trichoderma seed treatment also ameliorates physiological stresses such as ageing and seed dormancy (Delgado-Sanchez et al., 2010, 2011; Mastouri et al., 2010). The amelioration of oxidative stress has been suggested to be one of the major mechanisms of Trichoderma-mediated mitigation of abiotic stresses, e.g. addition of glutathione also had similar effects. Indeed, the expression of a Trichoderma glutathione S-transferase (GST) in tobacco enhanced tolerance to heavy metals and anthracene (a polycyclic aromatic hydrocarbon) through countering the oxidative stresses generated (Dixit et al., 2011a,b). Interestingly, the endochitinase gene that was originally transferred to plants for the control of plant pathogens was also reported to be imparting abiotic stress tolerance, owing to the action of release of elicitors (de las Mercedes Dana et al., 2006; Distefano et al., 2008). The beneficial effects of Trichoderma genes in plants have been reviewed recently (Hermosa et al., 2012).

In addition to the control of plant pathogenic fungi and bacteria, Trichoderma spp. have also been reported to be parasitic on nematodes, thus opening a new frontier in Trichoderma research (see Chapter 14, this volume; Sharon et al., 2001; Szabó et al., 2012). Because many plant pathogenic nematodes and fungi ‘collaborate’ in causing plant diseases (e.g. nematode-assisted penetration of the wilt pathogen Fusarium), a Trichoderma strain or a combination of strains that are effective against both fungi and nematodes might prove to be very effective in managing these complex diseases, which are otherwise difficult to control.

Viridiol, a secondary metabolite of Trichoderma virens, was reported earlier as having herbicidal properties (Howell, 1984; Jones et al., 1988; Hutchinson, 1999). The interest in this field has been renewed recently with the demonstration that the metabolites from some other Trichoderma spp. have herbicidal properties too (Javaid and Ali, 2011). This is one area that has not been explored rigorously, probably because of the availability of typical phytopathogens as more effective bioherbicides.

Trichoderma spp. have also negatively impacted agriculture, with several reports on the ‘green mould’ epidemics that caused havoc in mushroom cultivation. This is not unexpected though, because Trichoderma spp. are vigorous competitors and hence could easily infest the compost, and, because they are mycoparasites, the mushroom fungi could also be targets of parasitism. Interestingly, however, a species specificity seems to be there that restricts the epidemics to only certain species (Samuels et al., 2002; Hatvani et al., 2007; Komoń-Zelazowska et al., 2007).

1.3 Trichoderma in Industry

An interesting chance discovery that changed the face of the enzyme industry happened during the Second World War: a filamentous fungus thriving in its tropical home of the Solomon Islands made a crucial mistake. It happily degraded tents and uniforms of the military, which happened to be using its habitat. Initially, research on the organisms involved focusing on preventing the damage done by the hydrolytic enzymes of the fungi. But soon, numerous applications, especially for cellulases, boosted research on the degradation of plant material and nowadays this fungus, together with other species of the genus, offers a complete toolbox from plant protection to industrial fermentation for the production of enzymes and chemicals, including biofuels (see Chapter 13, this volume). It took some time, however, from discovery to industrial application, and, in the early days, many fungi were evaluated for the potential to degrade plant cell wall material along with Trichoderma reesei (Siu and Reese, 1953). At first, T. reesei (then called Trichoderma viride) was not even among the isolates studied for efficient cellulase production. Its efficient enzyme system later resulted in T. reesei becoming a model system for plant cell wall degradation. Since then, this fungus has undergone countless rounds of mutation by chemicals and radiation, along with improvement by genetic engineering (Seiboth et al., 2011). Having become one of the most prolific producers of plant cell wall degrading enzymes, it is now grown in liquid culture in huge steel fermenters, instead of the humid and rich tropical soil to which it was adapted, degrading the leftovers of plant growth and keeping the carbon cycle in balance.

With the advent of the biofuel industry, the interest in T. reesei has been renewed and is being actively investigated to improve its potential to degrade plant-based wastes.

Thanks to its strong cellulase promoters and the versatile tools developed for genetic engineering of this fungus (Kubicek et al., 2009; Schuster et al., 2012), T. reesei is also used as a host for heterologous protein production. A proof-of-principle study recently revealed the possibility of using T. reesei as a cell factory for the production of valuable chemical compounds (see Chapter 16, this volume). Another...