Antagonistic fungi, Trichoderma spp.: Panoply of biological control

Biochemical Engineering Journal 37 (2007) 1–20

Review

Antagonistic fungi, Trichoderma spp.: Panoply of biological control Mausam Verma a , Satinder K. Brar a , R.D. Tyagi a,∗ , R.Y. Surampalli b , J.R. Val´ero a a

INRS-ETE, Universit´e du Qu´ebec, 490, Rue de la Couronne, Qu´ebec, Canada G1K 9A9 b US EPA, P.O. Box-17-2141, Kansas City, Kansas, KS 66117, United States

Received 31 October 2005; received in revised form 6 May 2007; accepted 18 May 2007

Abstract Trichoderma spp. have been widely used as antagonistic fungal agents against several pests as well as plant growth enhancers. Faster metabolic rates, anti-microbial metabolites, and physiological conformation are key factors which chiefly contribute to antagonism of these fungi. Mycoparasitism, spatial and nutrient competition, antibiosis by enzymes and secondary metabolites, and induction of plant defence system are typical biocontrol actions of these fungi. On the other hand, Trichoderma spp. have also been used in a wide range of commercial enzyme productions, namely, cellulases, hemicellulases, proteases, and ␤-1,3-glucanase. Information on the classification of the genus, Trichoderma, mechanisms of antagonism and role in plant growth promotion has been well documented. However, fast paced current research in this field should be carefully updated for the fool-proof commercialization of the fungi. The aim of this review is to sum up the BCA activity potential of these fungi and to shed light on commercial production processes. In this regard, this review focuses on Trichoderma spp. discussing different aspects—pest control, growth promotion, bioremediation, production processes and market values. Nevertheless, more research and review of the information regarding these biocontrol agents are needed to exploit their actual potential, which is the salient objective of this review. © 2007 Elsevier B.V. All rights reserved. Keywords: Antagonism; Biocontrol agents; Microbial propagules; Trichoderma spp.; Wastewater; Wastewater sludge

Contents 1.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Existence of biological control agents (BCAs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Fungal BCAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3. Status of Trichoderma spp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4. Constraints in commercialization of Trichoderma spp. BCAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pest control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1. Trichoderma spp. as biofungicides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2. Modes of action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.3. Application in wood preservation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.4. Application in agriculture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. Limited application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2. Potential—future application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Invertebrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.1. Application potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.2. Rhizosphere—nematodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.3. Foliar application—aphids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Weeds. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Corresponding author. E-mail address: [email protected] (R.D. Tyagi).

1369-703X/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2007.05.012

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M. Verma et al. / Biochemical Engineering Journal 37 (2007) 1–20

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6. 7.

Growth promotion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Direct effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.1. Metabolite production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.2. Participation with ectomycorrhizal sphere in growth promotion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Indirect effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1. Induction of plant defence mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bioremediation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Microbial propagules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1.1. Spore/conidia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1.2. Mycelium and chlamydospore . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Mass scale production strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.1. Solid-state fermentation (SSF), and liquid fermentation (LF) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.2. Combined process. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Solutions to improve LF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.1. Inoculum effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.2. C:N ratio and level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.3. Nature of C and N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.4. Sporulation inducer compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.5. Physiochemical production parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.6. Fermentation modes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Selection of raw materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.1. Conventional/semi-synthetic substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.2. Alternate/recycled substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.3. Wastewater and wastewater sludge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5. Standard bioassay for quality control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6. Trichoderma spp. based formulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.7. Integrated pest management with Trichoderma spp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Market potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction 1.1. Existence of biological control agents (BCAs) Phytopathogenic microorganisms and insects have been coexisting with plants since the very beginning of agricultural evolution. Despite being a natural phenomenon, their mutual existence has adversely affected agriculture and forests from time to time. With the advent of technology, physico-chemical methods have been adopted to mitigate the phytopathogenic impacts on agriculture and forests. Use of crude (ash, raw extract of certain plants, lime) and chemical pesticides, physical traps for insects [1] are examples of these control methods. However, later on, integration of biological control with pre-existing methods has further revolutionized the agricultural and forest pest management. 1.2. Fungal BCAs Currently, the role of BCAs is a well established fact and has become increasingly crucial, and in several cases, complementary or even replacing the chemical counterparts where antagonistic fungi play an important part [2–4]. Fungal based BCAs have gained wide acceptance next to bacteria (mainly, Bacillus thuringiensis), primarily because of their broader spec-

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trum in terms of disease control and production yield [5]. In this context, Trichoderma spp. have been the cynosure of many researchers who have been contributing to biological control pursuit through use of fungi [6–17]. Furthermore, Trichoderma spp. share almost 50% of fungal BCAs market, mostly as soil/growth enhancers and this makes them interesting candidates to investigate [4]. 1.3. Status of Trichoderma spp. Although, Trichoderma spp. have been known for a long time (since 1865 [18]), the taxonomy and species identification were vague until around 1969 [19]. In fact, Druzhinina and Kubicek [20] have extensively reviewed species concepts and biodiversity in Trichoderma fungi. The authors have mentioned that Trichoderma fungi are difficult to distinct morphologically, however, the phylogenetic classification has rapidly reached 100 [21], and it is expected to increase consistently. In this context, the advancements as well as limitations of modern methods like genealogical concordance phylogenetic species recognition (GCPSR) and DNA-barcode system for safe identification of Trichoderma spp. warrant future investigations. The GCPSR requires the analysis of trees of several unlinked genes, whereas, DNA-barcode system is based on the defined nucleotide sequence differences of different Trichoderma spp.

M. Verma et al. / Biochemical Engineering Journal 37 (2007) 1–20

Nevertheless, application of Trichoderma spp. as BCAs in the environment as well as the reported epidemics of commercially grown mushroom (Agaricus bisporus) [22] and harmful effects on immunocompromised mammals [23] are reasons which warrant efficient and reliable species identification for Trichoderma fungi. The widespread application of Trichoderma spp. as BCAs has been exploited and reported only lately against several soil-borne phytopathogenic fungi [24,25]. Akin to most fungal BCAs, Trichoderma spp. can be efficiently used as spores (especially, conidia), which are more tolerant to adverse environmental conditions during product formulation and field use, in contrast to their mycelial and chlamydospore forms as microbial propagules [26]. Nevertheless, the presence of a mycelial mass is also a key component for the production of antagonistic metabolites [7,27]. Conidia and mycelia can be produced in either solid-state or liquid fermentation. In general, liquid fermentation is more suitable method over solid-state fermentation for large scale production, still special techniques are required for abundant conidia production. Trichoderma fungi are well known for their antagonism against several soil-phytopathogens, involving fungi, invertebrates, and bacteria (Table 1). Their BCA activity is mainly attributable to various anti-microbial/antagonistic compounds they produce, in addition to their aggressive mode of growth and physiology. Full exploitation of the BCA potential of Trichoderma spp. could easily provide growth enhancement of domestic plants, green house plants, and agricultural crops. 1.4. Constraints in commercialization of Trichoderma spp. BCAs Trichoderma spp. are also preferred in bioremediation due to the production of metabolites that are rich in peroxidases, and laccase enzymes [28,29]. Despite all the acquired understanding about antagonistic action and growth promotion of the Trichoderma spp., there are nevertheless some hurdles to their widespread success: (a) most of Trichoderma spp. based BCAs are unregistered and are being marketed simply as “soil enhancers”, probably due to lack of “well defined” modes of action of these fungi and their underdeveloped bioassay methods (to ensure product quality) [4]. Furthermore, the registration of Trichoderma spp. based BCAs as fungicides and growth promoters is time-consuming, expensive and frequently without well defined protocols; (b) raw materials like, glucose, sucrose, corn steep liquor, wheat bran, soya meal, fish meal, used in culture media for production of the fungi are very costly [30,31]; (c) low efficacy; (d) low spore yield; (e) difficulties in quantification of BCA activity. This has encouraged many researchers to investigate agricultural wastes [32], industrial wastes [13], and municipal wastes [33] as probable substrates for Trichoderma spp. production. To recapitulate, the essential information regarding Trichoderma spp. as BCA is scarce as most of the current literature is focused mainly on its commercial enzyme production capacity. The aim of this review is to sum up the BCA activity potential of these fungi and to shed light on commercial production pro-

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cesses. In this regard, this review focuses on Trichoderma spp. discussing different aspects—pest control, growth promotion, bioremediation, production processes and market values. 2. Pest control 2.1. Fungi 2.1.1. Trichoderma spp. as biofungicides In general, the current literature indicates that Trichoderma spp. have been mostly used as biofungicide agents (Table 1). First report on the subject may be credited to Coley-Smith et al. [34] who by means of microtome sections have shown that medulla of infected sclerotia of Sclerotium delphinii were completely replaced by hyphae and chlamydospores of Trichoderma hamatum on agar plates. Likewise, Henis et al. reported mycoparasitism (penetration and infection) of Trichoderma spp. against Sclerotium rolfsii, where chlamydospores were abundantly produced in contrast to conidia within the infected fungal sclerotia [35]. 2.1.2. Modes of action 2.1.2.1. Mycoparasitism. According to Punja and Utkhede [36], Trichoderma spp. are the most widely studied mycoparasitic fungi. However, their mycoparasitism is difficult to demonstrate in situ until very recently due to technical difficulties in making in situ microscopic observations (e.g., fluorescence imaging and differential staining), such as at the soil–root interface. Moreover, techniques involving antibodies, such as combined baiting-ELISA (enzyme linked immunosorbant assay) techniques to detect Trichoderma spp. in composts, would certainly increase our understanding of the mycoparasitic interaction of these fungi [37]. Previously, Cook [38] classified the mycoparasitic interactions as: (1) replacement (unilateral antagonism), (2) deadlock (mutual antagonism), and (3) intermingling (no antagonism), with lack of explanation at microscopic level. However, more recently the understanding of mycoparasitism has considerably improved [7,14,27,39–44]. Interestingly, the studies were carried out at genetic [44,45] and microscopic [27,43] levels. However, a broader concept concerning living plants (with the exception of preservation of wood, where Trichoderma spp. alone kill plant pathogenic fungi; discussed later), would be that after being treated with mycoparasites, plants induce defence mechanisms on their own. Further, this phenomenon leads to production of fungal inhibitory compounds by plants in addition to Trichoderma spp., thereby, facilitating mycoparasitism. Moreover, the mycoparasitism shown by Trichoderma sp. was host specific (e.g., Pythium oligandrum) [43]. Recently, the role of extracellular enzymes has been well documented by several researchers (e.g., proteolytic enzymes [46,47]; ␤-1,3glucanolytic system [48–50]; chitinase [44,51]). The complex group of extracellular enzymes have been reported to be a key factor in pathogen cell wall lysis during mycoparasitism. 2.1.2.2. Antibiosis. Antibiosis is the process of secretion of anti-microbial compounds by antagonist fungi to suppress

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Table 1 List of different Trichoderma spp. and respective BCA facts Active agent

Antagonist against

Responsible metabolites/factors

Disease/epidemic control

Fungi T. harzianum 1051, T. harzianum 39.1

Crinipellis perniciosa

Chitinase, N-acetylglucosaminidase, ␤-1,3-glucanase, total cellulase, endoglucanase, aryl-␤-glucosidase, ␤-glucosidase, protease and amylase Unknown inhibitory substances; extracellular metabolites or antibiotics, or lytic enzyme action

Witches’ broom disease (Crinipellis perniciosus) of Cocoa [63]

Lipolytic, proteolytic, pectinolytic and cellulolytic enzymes. Unknown (mycotoxins) antibiotic compounds (e.g., peptides, cyclic polypeptides) ␤-1,3-Glucanase and chitinase Competitive inhibition Antibiotic; anthraquinones Endo-chitinase Plant phytoalexin induction by antibiotic compound, gliovirin Volatile organic compounds, lytic enzymes and soluble antibiotics; nutrient competition

Fungal—seed-associated [10,11]

Rhizoctonia solani

T. harzianum, BAFC 742 T. sp. T. harzianum 25, T. viride T. harzianum T. virens “Q” strain

Sclerotinia sclerotiorum, BAFC 2232 Sclerotium rolfsii Serpula lacrymans Alternaria alternata Rhizopus oryzae/Pythium sp.

T. viride isolate, T60

Soft rot and basidiomycetes decay fungi, namely, Coniophora puteana, Postia placenta and Serpula lacrymans Rhizoctonia solani, Pythium ultimum, and Meloidogyne incognita Pyrenophora tritici-repentis (Died) Drechs. (anamorph = Drechslera tritici-repentis (Died) Shoem. Colletotrichum truncatum

T. virens isolates GL3 and GL21; T. harzianum T-203 T. harzianum, T. aureoviride, T. koningii

T. viride

Aspergillus flavus and Fusarium moniliforme

T. aureoviride T122, T. harzianum T66 Fusarium, Pythium, and Rhizoctonia strains and T334, and T. viride T124 and T228 T. viride, T. pseudokoningii and T. koningii Sclerotium cepivorum T. harzianum

Fusarium udum

T. harzianum T. virens and T. harzianum T. harzianum, T. koningii

Penicillium expansum Rhizoctonia solani Fusarium culmorum, Botrytis cinerea and Rhizoctonia solani

T. koningii, T. aureoviride, T. longibrachiatum T. harzianum

Sclerotinia sclerotiorum

T. harzianum and T. viride

Lasiodiplodia theobroma; Diplodia natalensis; Botryodiplodia theobromae; Fusarium moniliforme var subglutinans; Penicillium oxalicum Currie; Penicillium sclerotienum Yamamoto; Aspergillus niger van Tiegh; Aspergillus tamarii Kita; Rhizoctonia sp.

Alternaria alternata

Antibiotics gliovirin and gliotoxin, and other inhibitory metabolites Lytic enzymes such as chitinases and proteases; antibiotics; mycoparasitism Volatile compounds and non-volatile antibiotics, viridin with anti-fungal and anti-bacterial properties ␤-Glucosidase, cellobiohydrolase; ␤-xylosidase and protease enzymes Volatile organic compounds, lytic enzymes and soluble antibiotics Lytic enzymes such as chitinases and proteases; antibiotics Enzymes; antibiotics; mycoparastitism Enzymes; antibiotics Anti-fungal compound: 6-n-pentyl-2H-pyran-2-one (6PAP); lytic enzymes, mainly, chitinases, ␤-1,3-glucanase, and proteases Antagonism—enzymes; antibiotics and mycoparasitism Fungal cell wall degrading enzymes

Anti-fungal metabolites; non-volatile and volatile antibiotics

Damping-off of bean [8,142,160,161]

Fungal—soybean plant [55] Rotting of common vegetables [162] Fungal wood decay [70] Fungal plant disease [140] Cotton seedling disease [69,163,164] Sap stain discoloration of pine and spruce trees [165,166] Damping-off of cucumber [14,65,167] Tan spot and leaf blotch of wheat [85,168,169]

Brown blotch disease of cowpea [84] General fungal plant disease [108,170–172] Allium (onion seedlings) white rot [141,173,174] Pigeonpea wilt [175,176] Apple blue and gray mold [80,81] Stem canker or black scurf of potato [64] Plant pathogens [41,47,83]

Sunflower head rot [86] Fungal infection of leaves, stems, flowers and fruits of annual plants, especially vegetables and ornamentals [140] Fruit rot/wilt of banana; mango stem-end rot and post-harvest rotting of yams [78,79,177,178]

M. Verma et al. / Biochemical Engineering Journal 37 (2007) 1–20

T. lignorum, T. virens, T. hamatum, T. harzianum and T. pseudokoningii (Rifai) T. viride, T. harzianum

T. asperellum

Fusarium oxysporum

T. harzianum

Aphanomyces cochlioides, Rhizoctonia solani, Phoma betae, Acremonium cucurbitacearum, and Fusarium oxysporum f. sp. radicis-lycopersici Botrytis cinerea and Mucor piriformis

T. harzianum Bacteria T. viride T. hamatum

Invertebrates (insects and nematodes) T. viride, T. koningii, T. longibrachiatum, T. hamatum, T. harzianum T. harzianum Rifai ITEM 908 and ITEM 910 Trichoderma spp. Plants (weeds) T. virens (=Gliocladium virens) T. viride T. harzianum T. harzianum T. koningii T. virens a

Wilt of tomato [179]

Antagonistic factors like enzymes and antibiotics

Post-harvest rotting of strawberries [82]

Heptelidic acid—antibiotic action Antibiotic activity of an isocyanide metabolite—3-(3-isocyanocyclopent-2-enylidene) propionic acid

[90,91] Control of rumen bacteria [89]

Non-volatile and volatile antibiotics Unknown Gliotoxin, dimethylgliotoxin, heptelidic acid, viridiol and viridin

Post-harvest rotting of yams [177] Bacterial canker [87] Biocontrol of plant diseases [67]

Common plant fungal diseases [180]

Atta cephalotesa (a leaf-cutting, fungus growing ant) 1,3-␤-Glucanase, chitinases, proteases, and lipases. Unknown antibiotic compounds Schizaphis graminum (aphid) Polysaccharide lyases, proteases, and lipases

Damage to plant leaves by ants [15,181]

Caenorhabditis elegans; Meloidogyne incognita

Enzymes; antibiotics

Root-knot nematode of horticultural crops [100]

Setaria viridis and Amaranthus retroflexus

Phytotoxins, including viridiol, gliovirin, gliotoxin, and viridian; allelochemicals Phytotoxins and lytic enzymes

Broadleaf and grass weeds [16,17,66,163]

Antibiosis; enzymes Unknown Anti-fungal compounds Induction of terpenoids

Rotting of Capsicum annuum roots [155,182,183] Potato disease control [184,185] Protect wheat against take-all disease [186,187] Verticillium wilt of cotton [185]

Lantana camara; Rottboellia cochinchinensis; Mikania micrantha Phytophthora capsici; Phytophthora erythroseptica Verticillium dahliae Gaeumannomyces graminis var. tritici V. dahliae

Indirect antagonism: Trichoderma spp. inhibits the symbiotic fungus of Atta cephalotes, thereby, affecting the normal growth of the insect.

Poisoning of cereal crops

Invasive weed [102]

M. Verma et al. / Biochemical Engineering Journal 37 (2007) 1–20

T. viride T. harzianum (Rootshield® ) T. virens, T. koningii

Anaerobic bacteria—Bacteroides fragilis Rumen bacteria—Escherichia coli, Megasphaera elsdenii, Streptococcus bovis, Bacteroides ruminicola, B. succinogenes, Succinivibrio dextrinosolvens, Ruminococcus albus, R. flavefaciens Serratia sp. Clavibacter michiganensis Bacillus subtilis

Antibiosis, mycoparasitism and competition for nutrients Enzymes; antibiotics and anti-fungal properties

5

6

M. Verma et al. / Biochemical Engineering Journal 37 (2007) 1–20

and/or kill pathogenic fungi in the vicinity of its growth area [52]. Menendez and Godeas [53] reported a biocontrol study of Trichoderma harzianum against Sclerotinia sclerotiorum—a soil-borne plant pathogen attacking many economically important crops, such as, soybean. The authors studied antibiosis of T. harzianum against the plant pathogen, assuming that the beneficial effect was due to concurrent mycoparasitism and competition as reported earlier in a similar study [54–56]. In another example, despite close contact between hyphae of Trichoderma spp. and Fusarium moniliforme/Aspergillus flavus on co-culturing, hyphal penetration was absent, suggesting that mycoparasitism was not the sole cause for the observed inhibitory effects [11]. Therefore, metabolites produced by Trichoderma spp. (e.g. volatiles, extracellular enzymes and/or antibiotics) were considered to be the probable elements involved in antibiosis. Trichoderma spp. have been also effective in cases of a wide host range and in hindering the longevity of sclerotia of pathogenic fungi. Recently, Szekeres et al. [57] have reviewed antagonistic metabolites produced by Trichoderma spp. The metabolites are linear, amphipathic polypeptides, namely, peptaibols and peptaibiotics. They also discussed the physico-chemical and biological properties of these antibiotic compounds which included the disruption of lipid membranes, anti-microbial activities, and induction of plant resistance. 2.1.2.3. Competition. Celar [58] conducted a study on the forms of nutrients commonly available to phytopathogenic and antagonistic fungi. Earlier study reconfirmed the findings of Blakeman [59] that shortage of easily accessible nutrients for microorganisms, especially of those living in soil and on plant surfaces, could result in explicit nutrient competition among microorganisms [60,61]. In addition, Trichoderma spp. could compete and sequester ions of iron (the ions are essential for the plant pathogen, Serpula lacrymans as part of a non-enzymatic complex) by releasing compounds known as siderophores [62]. Thus, the cited examples confirmed that significance of competition for nutrients between Trichoderma and pathogenic fungi. Several authors have highlighted the significance of lytic enzymes in BCA activity and studied isolates of Trichoderma spp. with cellulose and chitin degradation characteristics [63–65]. Hutchinson [66] and Hanson and Howell [67] have reported the significance of secondary metabolites (antibiotic activity) in antagonistic action of Trichoderma spp. against pathogenic fungi Pythium ultimum and Rhizoctonia solani. However, there seems to be a general consent on the combined synergistic effect of the two factors (enzymes and antibiotic compounds) [52,68,69]. 2.1.3. Application in wood preservation 2.1.3.1. Hardwood. Biological control studies to protect wooden distribution poles by Trichoderma spp. have also been carried out against the dry rot fungus, S. lacrymans [70] and brown rot fungi, namely, Antrodia carbonica and Neolentinus lepideus [71]. From these studies, it was concluded that although Trichoderma spp. displayed a killing action against these fungi in

in vitro tests, yet their in situ action was inefficacious as observed earlier [72]. However, further studies by Bruce et al. [73] showed that in situ BCA potency of Trichoderma spp. was still effective. In cases of lesser efficacy, a non-uniform distribution or impoverishment of Trichoderma spp. and compartmentalization of pathogenic fungi could be involved. Therefore, suitable in situ application techniques should be of prime importance, for a successful field application of BCAs. Meanwhile, Score and Palfreyman [70] postulated that the presence of varying nutrient concentrations in wood might have a marked effect on the nature of antagonistic interactions. They also showed that varying nutrients in enriched media produced a clear effect on hyphal extension rate of several Trichoderma spp. affecting their BCA activity. However, determination of actual BCA activity would require extensive studies on wood like, the possible interactions of various Trichoderma spp. metabolites with major wood components, namely, tannin, and lignin. 2.1.3.2. Cosmetic woods. Ejechi [12] investigated the ability of Trichoderma viride to inhibit the decay of obeche (Triplochiton sceleroxylon) wood by the decay fungi Gloeophyllum sp. and G. sepiarium under field conditions. The study was conducted over a period of 11 months, covering dry and wet season in tropical environment. T. viride exhibited total inhibition of the decay fungi by means of mycoparasitism and competition for nutrients. Trichoderma spp. are currently the most extensively investigated biocontrol fungi for forest product preservation, and, on a number of occasions, have successfully provided more effective protection against certain wood decay fungi in comparison to other antagonistic fungi, e.g., Penicillium sp. [71,74]. Using modified versions of American [75] and European [76] standard test methods as well as a soil burial test system, Tucker et al. [77] have shown that certain isolates of Trichoderma spp. were totally effective in protecting wood against certain basidiomycetes. 2.1.4. Application in agriculture 2.1.4.1. Fruits and vegetables. Several Trichoderma spp. have also been used to protect commercially important fruits and vegetables, such as banana, apple, strawberries, mango, potato, and tomato during post-harvest storage (Table 1). Mortuza and Ilag [78] utilized 10 isolates of Trichoderma spp. including T. harzianum and T. viride against the banana fruit rot pathogen, Lasiodiplodia theobromae. Their investigation confirmed that mycoparasitism was associated with antibiosis and competition for substrate. The authors compared the cultural filtrates of Trichoderma spp. with a chemical fungicide, namely, BenomylTM , and concluded that Trichoderma spp. based fungicide could not be totally as effective as their chemical counterparts. However, the authors underestimated the role of Trichoderma spp. based formulations for field application, which might substantially enhance the BCA activity. Biological control of mango stem-end rot using T. viride was studied by Moreno and Paningbatan [79], where they reported mycoparasitism and antagonism as major BCA activity factors. Batta [80,81] examined the invert-emulsion formulation of T. harzianum Rifai against blue mold infection of apple to control post-harvest fruit decay. The author attributed the BCA activity

M. Verma et al. / Biochemical Engineering Journal 37 (2007) 1–20

7

Fig. 1. Growth promotional activities of Trichoderma spp. Indirect: (a) mycoparasitism, (b) competition; direct: (c) mycelial growth around plant rhizosphere and production of metabolites.

of Trichoderma spp. to the necessary period of conidial humectation in order to germinate and penetrate into the plant pathogenic fungi. It was also reported that the invert-emulsion was better application mode for fungal BCAs like Trichoderma spp. Brewer and Larkin [64] reported Trichoderma spp. to be potential antagonists for stem disease of potato in comparison to several other fungal BCAs. On the other hand, Hjeljord et al. [82] reported that application of Trichoderma spp. on greenhouse strawberries could control post-harvest rotting. They emphasized that temperature and nutrients influenced the BCA activity of T. harzianum. Results of Cooney and Lauren [83] confirmed antagonistic metabolite production by a Trichoderma sp. induced by the presence of pathogenic fungi (300–700% increase in metabolite production in the presence of plant pathogenic fungi). 2.1.4.2. Crops/seeds. Trichoderma spp. are well-recognized fungal antagonists of crops/seeds pathogens. Biocontrol of brown blotch of cowpea caused by Colletotrichum truncatum by pre-treatment of cowpea seeds in T. viride spore suspension was considered to be due to both mycoparasitism and antibiosis [84]. Similarly, the application of Trichoderma spp. for the control of wheat [85] and sunflower [86] fungal diseases has been

based on their mycoparasitic and antibiotic activities. However, particular factors triggering antagonism (molecular signal for mycoparasitism, as Trichoderma spp. were able to recognize their specific host) were not available. In another instance, the beneficial action of T. virens for pre-treatment of cotton seedlings has been reported. It was demonstrated that the induction of plant defence system and the suppression of pathogen germination by antagonistic compounds produced by germinating cotton seedlings were the dominant biocontrol mechanisms [69]. Thus, it is evident that the processes of mycoparasitism, antibiosis, and competition for substrates are well documented, as exemplified in Fig. 1. However, the exact phenomena involved in the overall antagonistic action of Trichoderma spp. is still not well understood. It is postulated that Trichoderma spp. initially succeed in antagonism via hyphal interactions, probable primal step in antagonism. Later, the antagonistic fungi kill the phytopathogenic fungi by means of toxins and consume them using a combination of lysozymes [4]. On the other hand, the fungicidal activity of Trichoderma spp. is well known with respect to most of the fungal phytopathogens. Most researchers agree to the fact that the antagonists (Trichoderma spp.) control pathogens via interlinked synergistic

8

M. Verma et al. / Biochemical Engineering Journal 37 (2007) 1–20

complex strategies of mycoparasitism, antibiosis, and competition. Therefore, in order to exploit maximum potential of Trichoderma spp. against fungal pathogens, factors (e.g., distribution of inoculum at infected sites, concentration of inoculum, specificity towards pathogens, environmental conditions and enrichment medium—if any) affecting one or many of the antagonistic strategies should be optimized. 2.2. Bacteria 2.2.1. Limited application In contrast to other fungi, Trichoderma spp. have been reported to have limited applications in biocontrol of pathogenic bacteria. An immediate explanation would be that bacteria generally have a faster metabolic rate than fungi. Thus, antagonism via physical interaction such as, mycoparasitism would be too slow to be effective from BCA point of view, where faster action is a must. However, if the formulated metabolites from Trichoderma spp. were considered, the BCA potential of antagonist fungi would be considerably higher. 2.2.2. Potential—future application In view of limited studies on anti-bacterial action of Trichoderma spp., very few examples have been reported in the literature, which have been listed in Table 1. Altogether, antibacterial action of Trichoderma spp. was based only on the action of antibiotic compounds produced and no physical interaction between antagonist and pathogen was mentioned [67,87–91]. Therefore, in the present scenario, despite Trichoderma spp. possessing anti-bacterial potential, their applicability cannot be advocated and actually employed in field set-ups. 2.3. Invertebrates 2.3.1. Application potential Trichoderma spp. are basically soil-borne saprophytic fungi which possess a symbiotic relationship with the plant rhizosphere [92]. In addition, they have innate ability to produce several chitin (a major component of cellular structure in invertebrates) degrading enzymes (endochitinases and exochitinases) in order to survive on and/or antagonize pathogen organism [49,51,63,93]. 2.3.2. Rhizosphere—nematodes Nematodes are mostly present in rhizosphere than in the bulk soil, therefore, their antagonist(s) should also be in sync with rhizosphere. Fungi like Trichoderma spp. fit very well into this category [94–100]. Most studies on nematodes concurred that the promising fungal antagonists—Trichoderma spp., had different and in fact multiple modes of action. For example, Trichoderma virens invaded, ramified, grooved and vacuolated the root-knot nematode eggs. Eapen et al. [100] reported easy staining of eggs for microscopy due to the increased permeability of eggshell. The antagonistic action of Trichoderma spp. was chiefly attributed to chitinolytic activity of the fungi on cellular structure of nematodes, which is rich in chitin. Additionally, unlike bacteria,

nematodes were mainly antagonized by parasitism and antibiosis akin to fungal pathogens. 2.3.3. Foliar application—aphids The insecticidal activity of two strains of T. harzianum against aphids has been reported [101]. Since T. harzianum is currently considered as an important BCA candidate, its insecticidal activity has further importance. The authors proposed that the action of toxins produced by T. harzianum strains was facilitated by cuticle degrading extracellular enzymes (proteases and chitinases) that enabled the insertion of the toxins inside cuticle. Thus, many Trichoderma spp. are reported as occasional parasites of invertebrates. Therefore, the actual potential of these fungi in the natural suppression of insects affecting economical crops is still underrated. This furthermore warrants research on using Trichoderma spp. based BCAs to cope with insect related plant disorders. 2.4. Weeds Herbicidal action of Trichoderma spp. has also been reported by some authors ([16,17,66,102]; Table 1), however, weed control by using Trichoderma spp. is still a relatively unexplored field. Heraux et al. [16,17] have reported the weed control action of T. virens. The study reported that T. virens with and without combination of composted manure and rye cover crop controlled a mixed community of grass weeds and broadleaf weeds. The weed control action was comparable to chemical herbicides, namely, metribuzin, sethoxydim and ethalfluralin. T. virens composted chicken manure and rye cover crop has showed herbicidal activity [16,17,66]. The herbicidal activity was attributed to: (a) viridiol produced by T. virens and (b) the herbicidal molecules, namely, (3H)-benzoxazolinone (BOA) and 2,4-dihydroxy-1,4(2H)-benzoxazine-3-one (DIBOA) released during composting of chicken manure and rye cover crop [16,17]. However, there is a need for future studies to ensure reliable performance prior to commercial utilization of T. virens. In fact, utilization of Trichoderma as herbicides is a relatively new approach and is not feasible under present scenario. Nevertheless, future research should be carried out to increase the concentration of viridiol and potential phytotoxins (Table 1) produced during fermentation. Agricultural residues other than rye cover crop and chicken manure should also be examined in order to make the process economically feasible. 3. Growth promotion 3.1. Direct effect 3.1.1. Metabolite production Literature is replete with many studies on plant growth promotional activity of Trichoderma spp. [4,36,42,103]. Some researchers have suggested production of growth hormones [104,105] and enhanced transfer of minerals to rhizosphere [104] as direct factors governing spectacular performance of Trichoderma spp. based BCAs as also reported in Table 2. Besides, Trichoderma spp. have been reported to promote growth in

[194,195] [196] Co-metabolism in the presence of glycerol Co-metabolism in the presence of glucose 800–1100 mg PAHs/kg soil 100 ␮g/g soil Polyaromatic hydrocarbons (PAHs) Fluorene

[193] [58] Nitrogen and phosphorus NH4 + –N and NO3 − –N Chickpea Pine

[192] Cyanide to ammonia Cyanide

T. harzianum (O90, O77, O82 and O80) and one T. pseudokoningi strain (O10) T. harzianum, T. viride and T. virens T. longibrachiatum, T. harzianum, T. viride, and T. koningii Trichoderma sp. T. hamatum, T. harzianum and T. koningii

[106,191] Rhizosphere enhancers—N and P Strawberry, Fragaria ananassa T. harzianum DB11

[188–190]

Saprotrophic fungus, Trichoderma sp. mobilized N from organic compounds via ectomycorrhizal fungi incapable of directly uptaking organic N from soil Mycorrhizal fixation of phosphorus and nitrogen Co-metabolism of cyanide in the presence of glucose Nutrient supplementation Competitive inhibition NH4 + –N; organic N (amino acid) P. resinosa Ait (red pine) T. sp.

Bioremediation/Suggested mode of growth promotion Growth promoters/target compound concentration Plant name/target compound Trichoderma sp.

Table 2 Bioremediation and suggested plant growth mechanisms by different Trichoderma spp.

Reference(s)

M. Verma et al. / Biochemical Engineering Journal 37 (2007) 1–20

9

strawberries [106]. Despite all the research, there is a need for extensive optimization of application conditions and active field studies. 3.1.2. Participation with ectomycorrhizal sphere in growth promotion Lindsey and Baker [107] demonstrated that the symbiosis of T. viride in rhizosphere helped in growth of gnotobiotic (either, well defined or, no microflora present) tomato, thereby, showing growth promotional ability of this fungus. The physical presence of mycelial mass in rhizosphere in itself would serve as appendage to the normal rhizosphere of plants, thereby, enhancing nutrient uptake. Further, the advantages of Trichoderma spp. based BCAs for growth promotion are enhanced due to symbiosis with ectomycorrhizal sphere [16,108,109]. Trichoderma spp. easily acquire nutrients from complex substrates like, proteintannin, and glucosamine, in soil due to their ectomycorrhizal association. Further, the nutrients were easily utilized by the plant due to their mutual symbiotic relationship. In particular, Trichoderma spp. has been extensively utilized in waste composting [17,66,109], which ultimately ends up in agricultural land, consequently affecting plant yield. The positive role of Trichoderma spp. in ectomycorrhizal sphere has been also elaborated by Wu et al. [110] which is an indirect mode for their plant growth promotional activity. 3.2. Indirect effect 3.2.1. Induction of plant defence mechanism In addition to the well-recognized mycoparasitic nature of Trichoderma fungi, induction of resistance against pathogens in plants has also been reported [27,42,44,111] as indirect growth promotion factor. The authors suggested that association of Trichoderma with roots, and several lytic enzymes induced by plant defence system destroyed and consumed the pathogen cell wall. Finally, it provided nutrient to the plant as depicted in Fig. 1. A mycoparasitic interaction study in this context revealed that ech42 (a chitinolytic enzyme encoding gene) transcription was induced prior to physical contact of T. harzianum with host pathogen fungus [44]. The authors also expressed the possibility of different mechanisms of induction for each type of lytic enzyme and the necessity to characterize these enzymes and their relevance in mechanisms of biocontrol. Available literature demonstrates the plant growth promotional activity of Trichoderma spp., still exact or quantitative BCA assessment is difficult due to multiple factors associated and scarce information available. Nevertheless, there are many Trichoderma spp. based commercial products in market which aim at greenhouse plants (mainly ornamental and garden vegetables) as depicted in Table 3. 4. Bioremediation The concept of utilizing fungi for bioremediation of soil contaminated with certain pollutants is relatively older. There is ample evidence of various Trichoderma spp. contributing to polycyclic aromatic hydrocarbons (PAHs) degradation,

Israel Spain Mycontrol (EfA1) Ltd. [180] Trichoderma 2000 TUSAL® T. harzianum T. harzianum

5. Production 5.1. Microbial propagules

Source: http://www.google.com internet search engine.

India – Trieco T. viride

Ecosense Labs

whilst affecting native mycorrhizal fungi both positively and/or, negatively [112]. Saraswathy and Hallberg [113] reported a maximum of 75% removal for pyrene (4-ring PAH) at 50 mg l−1 for axenic cultures of Trichoderma spp. while pyrene served as sole carbon source. Katayama and Matsumura [28] demonstrated degradation potential of rhizosphere-competent fungus Trichoderma sp. against several synthetic dyes, pentachlorophenol, endosulfan, and dichlorodiphenyl trichloroethane (DDT). Thus, Trichoderma spp. have found application in herbicide/pesticide laden soil bioremediation as sustainable approach. Review of present literature suggests that hydrolases, peroxidases, laccases and other lytic enzymes produced in abundance by Trichoderma spp. are probable factors aiding in degradation of these contaminants. From BCA point of view, bioremediation potential of fungi like Trichoderma would be an additional advantage as this will aid in soil enhancement where excessive use of herbicides should to be curtailed. However, this can be achieved, if and only if the contaminated soil is inoculated with Trichoderma spp. at regular defined intervals. In fact, inclusion of Trichoderma spp. in “integrated pest management” program is a must. In the current context, the scientific community believes that use of herbicides cannot be significantly reduced due to immediate concern of needs of agricultural commodities. Therefore, consistent simultaneous application of some “detoxifying” agents along with (Trichoderma spp. versus harmful pesticides/herbicides; the former can tolerate and degrade application concentrations of several pesticides/herbicides) would provide an agreeable soil environment. This will assure not only the health of soil and plant, but also a sustained crop yield protection. Future research must be streamlined on consistent-simultaneous use of Trichoderma spp. as BCA cum soil remediation agent along with obligatory pesticides/ herbicides.

– –

U.S., U.S., Belgium –

T. harzianum T. harzianum and T. polysporum T. harzianum and T. viride T. spp.

TrichopelTM , TrichojetTM , TrichodowelsTM , TrichosealTM PromotTM , Trichoderma 2000, Biofungus

J.H. Biotech, Mycontrol, Ltd., De Ceuster

Armillaria, Botryosphaeria and other fungal diseases Growth promoter, Rhizoctonia solani, Sclerotium rolfsii, Pythium spp., Fusarium spp. on nursery and field crops For management of Rhizoctonia spp., Pythium spp., Fusarium spp., root rot, seedling rot, collar rot, red rot, damping-off, Fusarium wilt on wide variety of crops Rhizoctonia solani, Sclerotium rolfsii, Pythium Agrimm Technologies

Antagonism by enzymes and antibiotics New Zealand

Israel U.K., Sweden Mycoparasite living on other fungi Mycoparasite Botrytis cinerea and others Tree-bound pathogens Makhteshim Bio-Innovation

U.S., Europe

RootShieldTM , BioTrek 22GTM , SupresivitTM , T-22GTM , T-22HBTM TrichodexTM BinabTM T. harzianum

BioWorks, Wilbur-Ellis, Borregaard

Parasite, competitor

Antagonist

Soil pathogens that cause damping off and root rot, esp. Rhizoctonia solani and Pythium spp. Soil pathogens—Pythium, Rhizoctonia, Verticillium, Sclerotium, and others Soil Guard Gliocladium virens#

Certis

Type of action as per manufacturer Manufacturers and suppliers

Pests controlled

12GTM

Trade name Beneficial organism

Table 3 List of Trichoderma fungi based BCAs and respective suppliers

U.S.

M. Verma et al. / Biochemical Engineering Journal 37 (2007) 1–20 Country registered

10

5.1.1. Spore/conidia The ultimate objective of any BCA lies in its feasibility of economical mass production which also holds true for Trichoderma spp. based BCAs. Further, from Table 3 and Fig. 2, it is obvious that Trichoderma spp. based BCAs are commercially viable as numerous commercial products exist in market, still a majority of them (not presented here) are “unsung” BCAs. A vast majority are rather being promoted as soil enhancer and/or growth promoter. Almost all available Trichoderma spp. based BCA products contain spores as active ingredients [4,36,80,81]. This could be attributed to the physiological aspects of their three microbial propagules, namely, mycelia, conidia, and chlamydospores [26,31,114]. The three propagules possess distinct physiological characteristics in terms of production, stability and BCA activity. Therefore, it is imperative to select the best suitable form of Trichoderma spp. propagules in order to efficiently execute their BCA action.

M. Verma et al. / Biochemical Engineering Journal 37 (2007) 1–20

11

aged major BCA producers to consider SSF as a viable mass scale option [118]. Unfortunately, accurate information regarding factors directing sporulation process in LF of fungi, especially, for Trichoderma spp. is scarce and incomplete, besides being proprietary. Therefore, production of spores in LF still remains a challenging task and warrants considerable research inputs.

Fig. 2. Trichoderma spp. based biofungicide market statistics. Other biofungicides include bacteria, nematodes and virus. Note: The market is based on scattered data of registered biofungicides.

5.1.2. Mycelium and chlamydospore Although, mycelia have excellent BCA activity, unfortunately, they cannot survive down stream processing steps such as drying and hence are not useful [26]. On the other hand, chlamydospores require a period of 2–3 weeks for cultivation and likewise could not survive drying processes [31], albeit, they are more stable than mycelia. Meanwhile, as said above, conidia are active as BCA, less susceptible to several environmental conditions and could be produced faster (3–4 days) in abundance [33]. Thus, production of Trichoderma spp. as conidia would be the best option from BCA application point of view. However, the presence of mycelia along with conidia in the production media cannot be ruled out. In addition, simultaneous mycelial production would insure presence of various essential metabolites (e.g., antibiotics) for BCA activity [66,67]. Thus, production of Trichoderma spp. containing conidia as main propagules along with mycelia could be the best production strategy.

5.2.2. Combined process To overcome limitations of SSF and LF many researchers also suggested hybrid strategies involving both SSF and LF. Normally, LF is followed by SSF in many industrial production processes. However, relevant information on production techniques is so far evasive in current literature. In a typical Trichoderma spp. production process, 2–3 days old broth of LF is used as inoculum for solid substrates, e.g., bran, rice, grain-husk and others. The solid substrates thus inoculated were incubated for further 2–8 days, followed by addition of formulation agents, e.g., carboxy methyl cellulose, silica, talc and moderate temperature (about 20–40 ◦ C) air-drying below 8–10% moisture content. However, labour-intensive nature of similar techniques certainly could not replace LF. Meanwhile, the problems in LF will no doubt eventually be overcome. 5.3. Solutions to improve LF

5.2. Mass scale production strategies

5.3.1. Inoculum effect The ongoing research also indicates that several techniques might help in improving sporulation during LF. For example, fungal morphology and the general course of fermentation has been primarily affected by the amount, type (spore or vegetative) and age of the inoculum [119–121]. In general, studies carried out on fungi until now suggested that high spore concentrations in inoculum (≤105 to 108 spores/ml) were not good for sporulation. Therefore, any direct role of inoculum (in form of spores) in sporulation could be ruled out. Nevertheless, same study also suggested that inoculum could be important for production of metabolite(s) and mycelial mass responsible for the BCA activity. Eventually, type, quantity, and age of inoculum should be explored for probable enhancement of sporulation.

5.2.1. Solid-state fermentation (SSF), and liquid fermentation (LF) Commercial success of Trichoderma spp. based BCAs would also require economically feasible mass scale production processes (as 35–40% costs of production depends on raw material). Of the two broad categories of production, namely, solidstate fermentation (SSF) and liquid fermentation (LF), LF has been adopted by many researchers despite lower sporulation [115,116]. Labour, scale-up, process control, productivity, material handling (pumping, pressurized lines), compatibility with pre-existing large scale facilities are some positive features of LF that encourage most researchers to pursue LF in lieu of SSF. However, SSF is often preferred to LF, when production scale is of moderate range and labour force is cheap [117]. Additionally, recent advancements in industrial automatization have encour-

5.3.2. C:N ratio and level Variation in values of carbon: nitrogen (C:N) ratio in medium could also be helpful in sporulation/conidiation of Trichoderma spp. Olsson et al. [122] observed start-time of sporulation phase in Trichoderma reesei Rut-30 and remarked that decreasing C:N ratio adversely affected sporulation. Moreover, studies on other fungi, namely, Neurospora crassa [123] and Beauveria sp. [124] also showed marked effect of C:N ratio on sporulation. Therefore, manipulation of C:N ratio remains a viable option for further investigation on sporulation process of Trichoderma spp. As mentioned earlier, the antagonism study of Trichoderma spp. by Score and Palfreyman [70] used a standard complex medium (malt extract agar, MEA) and a minimal essential medium (MEM) designed to mimic the C:N ratio in the pathogen

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infected wood. The findings revealed that Trichoderma spp. could be significantly antagonistic even at low nitrogen content. Therefore, any increase in C:N ratio owing to decrease in N would not affect the BCA activity of these fungi. In other words, substrates with higher C:N ratio could be beneficial for both sporulation and BCA activity. 5.3.3. Nature of C and N Sporulation of Trichoderma spp. is greatly affected by the nature of carbon and nitrogen sources used as substrates. Pascual et al. [125] observed that peptone, free amino acids (e.g., arginine), mannose, xylose, and fructose induced high level of sporulations. It could be inferred that exploring various carbon and nitrogen sources and their pre-treatments could be potential means to induce sporulation. 5.3.4. Sporulation inducer compounds Introduction of some triggering agents has also been helpful in sporulation process. These agents might be metal ions (e.g., manganese ions [126]), or complex organic compounds. Roncal et al. [127] have isolated conidiogenol and conidiogenone, tetracyclic diterpenes with potent and selective inducing activity for conidiogenisis in Penicillium cyclopium. These findings are still at their natal stage and might be helpful in practical application at later stage. Nevertheless, it appears that introduction of some metal ions and/or some complex compounds to the growth medium could induce sporulation. Perhaps, various metals and complex compounds in renewable waste, could be helpful in achieving high spore concentration. 5.3.5. Physiochemical production parameters Environmental parameters like, temperature have been less critical parameters for sporulation [128]. Meanwhile, pH of the medium plays an important role. Although, conidial fungi can grow over a wide range of pH, they grow and sporulate maximally near neutral pH [126]. Felse and Panda [129] investigated the effect of agitation conditions on a Trichoderma sp. and inferred that extremely agitated conditions were not good for growth and sporulation. Dissolved oxygen and dissolved carbon dioxide may also play an important role in inducing sporulation, probably by imposing mass transfer related stress conditions on the culture. The sporulation in fungi is also an important mode of reproduction, therefore, sporulation of Trichoderma spp. could also be correlated to the respiration quotient (RQ). 5.3.6. Fermentation modes In general, there are no research reports on the studies regarding culture conditions like continuous and fed-batch culture in fermenter that allow finer control of substrate concentration (solids concentration, C:N ratio), biomass (mycelia and/or spores concentration), single/multiple nutrient addition (feeding strategy for complex and/or simple substrates). Thus, the above listed parameters should be examined and studied adequately to induce sporulation.

5.4. Selection of raw materials 5.4.1. Conventional/semi-synthetic substrates Several growth/production media for Trichoderma spp. spores production are presented in Table 4. Use of media based on molasses, d-glucose, cellulose, or soluble starch have been also reported by many authors [31,115,116]. In general, maximum sporulation of the order of 108 spores/ml fermentation broth could be achieved. However, it should be noteworthy that production of Trichoderma spp. spores for BCA use (application in soil), using these substrates could not be economical, owing to high raw material cost and moderate sporulation. Meanwhile, a much higher spore concentration (≈107 to 1013 CFU/ha application requirements will necessitate > 107 to 1010 CFU/g formulated product, consequently requiring ≥107 to 1010 CFU/ml fermented medium) will be required for field application [82]. 5.4.2. Alternate/recycled substrates In order to obtain crude proteins, enzymes as mycelial mass, use of effluent of oil mill as substrate for producing Trichoderma spp. has been investigated, where the fermentation time was longer (≥2 weeks) [130] Felse and Panda [93,129] have reported the use of untreated crab shell to increase chitinase production. Olsson et al. [122] used cellulose, raw and treated sugar beet pulp without additional nutrients and Mendel’s medium components for the development of inoculum. Meanwhile, studies on steam-pretreated willow (lignocellulosic material) [32,131] suggested replacement of glucose and corn steep liquor in Tanaka media (Table 4) by Avicel (10 g l−1 ), or by corn fibre dry mass (20–140 g l−1 ). Several studies have been reported on the use of alternative cheap raw material for Trichoderma spp. production processes [132–135]. Remarkably, in the preceding examples, the authors studied cellulosic and hemicellulosic enzymes producer Trichoderma spp. In all studies, the BCA potential of fermented products was not the main objective of the authors. The sole presence of metabolites in Trichoderma spp. based BCA is not sufficient for its application viability, high spore concentration is equally important. In fact, most studies focused mainly on production of Trichoderma spp. for metabolites (enzymes and/or antibiotics) and not sporulation (an important factor for practical application of BCAs). Therefore, validation of commercial viability of these raw materials/processes from BCA point of view would require further investigations aimed at spore production and bioassay against commercial pests. 5.4.3. Wastewater and wastewater sludge Based on the preceding discussion for economical (low cost raw material) and efficient production (higher sporulation) of Trichoderma spp. based BCAs, a novel array of substrates have to be explored. Akin to prior mentioned wastes, experimenting with wastewater and wastewater sludges (source of carbon, nitrogen, phosphorus, and other essential nutrients for many microbial processes) could provide probable viable solution to combat the raw material cost and enhance sporulation, as in the case of B. thuringiensis [136]. In this context, Verma et

M. Verma et al. / Biochemical Engineering Journal 37 (2007) 1–20

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Table 4 Growth/production media used for the production of Trichoderma spp. for BCA use Media

Composition (g l−1 )

Basal salt medium

Yeast extract, 0.5; (NH4 )2 SO4 , 1; MgSO4 ·7H2 O, 0.3; KH2 PO4 , 1.36; either cellulose powder or the holocellulose, 10 Glucose, 10; urea, 0.3; (NH4 )2 SO4 , 1.4; KH2 PO4 , 2.0; CaC12 ·2H2 O, 0.4; MgSO4 ·7H2 O, 0.3; peptone, 0.75; yeast extract, 0.25; FeSO4 ·7H2 O, 0.005; MnSO4 ·4H2 O, 0.0016; ZnSO4 ·7H2 O, 0.0014; CoC12 ·6H2 O, 0.020 Glucose, 20; urea, 0.3; corn steep liquor, 10; (NH4 )2 SO4 , 1.4; KH2 PO4 , 2.0; CaC12 ·2H2 O, 0.4; MgSO4 ·7H2 O, 0.3; proteose peptone (Difco), 1; FeSO4 ·7H2 O, 0.005; MnSO4 ·4H2 O, 0.0016; ZnSO4 ·7H2 O, 0.0016; CoC12 ·6H2 O, 0.004 Glucose, 25; NaCl, 25; corn steep liquor, 5; molasses (blackstrap), 50. Sucrose, 20; NaNO3 , 6; KH2 PO4 , 1.5; MgSO4 ·7H2 O, 0.5; CaCl2 , 20. Glucose, 20; MgSO4 ·7H2 O, 1; KH2 PO4 , 2; ammonium tartrate, 1; FeSO4 , 0.001 Quartz sand, 1000; corn cobs, 400 (in 1600 ml liquid). Quartz sand, 1000; wheat bran, 400 (in 800 ml liquid). Quartz sand, 1000; cornmeal, 40 (in 200 ml liquid) Sucrose, 20.0; NaNO3 , 2.0; MgSO4 ·7H2 O, 0.5; KCl, 0.5; FeSO4 , 0.01; agar, 20.0 Potato starch; dextrose Oat flour, 30; agar, 15; ZnSO4 ·7H2 O, 0.01; CuSO4 ·5H2 O, 0.005 Vogel minimal salts medium and 1.5 g l−1 of EPS as carbon substrate Cranberry pomace, 10 g; CaCO3 , 0.5 g; water, 20 ml; NH4 NO3 , 0.5 g, and or fish protein hydrolysate (FPH), 2 ml (In w/v%) KH2 PO4 , 0.2; (NH4 )2 SO4 , 0.14; urea, 0.03; MgSO4 ·H2 O, 0.03; CaCl2 , 0.03; peptone, 0.1; crude cell wall of fungi, 1.0; plus trace metal solution (1.0 ml) Farm yard manure, rice chaffy grains, dried banana leaf, banana pseudostem and rice bran (500 g) and 100 ml of 30% molasses solution (v/v)

Mendel’s medium

Tanaka medium

M-CSL, SN, GT

Corn cobs, wheat bran, cornmeal

Czapek dox agar medium PDA Oat agar Botryosphaeran (exopolysaccharide (EPS)) Cranberry pomace-based medium

Crude cell wall preparations from barley

Organic substrate medium

Remarks (spore yield)

Reference

Semi-synthetic (LF) [spore yield ≈ 106 to 107 ml−1 ], pHa 4–7, temperaturea : 28–30 ◦ C; incubation timea : 4–21 days

[197]

[30]

[198]

[31]

Complex (SSF) (liquid; any of, M-CSL, SN, GT, or water) [spore yield ≈ 107 to 108 g−1 ]

[31]

Synthetic (SSF)

[134]

Semi-synthetic (SSF)

[122]

Semi-synthetic (LF)

[199]

Semi-synthetic (SSF), 5 mg/g pomace protein

[13]

Semi-synthetic (LF)

[200]

Complex (SSF) organic substrate

[178]

This table contains different media used for Trichoderma spp. BCA production. a Valid for all media types.

al. [33,137] have reported production of T. viride on municipal wastewater from BCA point of view. The studies advocated potential utilization of municipal wastewater sludge and industrial wastewaters as shown in the mass balance flow chart (Fig. 3) according to the proposed estimation of use of sludge. It was assumed for the schematic comparison between conventional and alternative routes that Trichoderma spp. fermented broths of conventional media/wastewaters/wastewater sludges will be mixed with either dry Talc/silica powder, or dry dewatered sludge powder as carrier material in the ratio of 1:99 (w/w), respectively. Eventually, for conventional route (conventional medium—108 CFU/ml) either extensive drying would be required or the process may not be viable to obtain conidia concentrations ≥107 CFU/g formulated powder. On the other hand, alternative route (wastewater sludges/wastewater sludges—107 to 1010 CFU/ml) would require moderate air drying. Therefore, for field application of fungal agents at a rate of 1013 CFU/ha [138] would warrant consumption of approxi-

mately 1–1000 kg/ha of dewatered sludge depending upon the selected raw material. Thus, Trichoderma spp. production on sludge would not just serve as potent BCA, but also as a novel technique for sustainable sludge management. These wastes often contain certain pollutants like, pesticides, metals complexes, whilst Trichoderma spp. are quite efficient in degrading these pollutants (e.g., organochlorine pesticides) [139]. They can tolerate high metal content by chelating metal ions [62]. The aforestated fact strengthens and encourages the ideology of using these wastes as substrates for Trichoderma spp. based BCAs. Furthermore, wastewater and wastewater sludges contain various complex compounds along with the essential components, hence there are ample opportunities that one could encounter compound(s) that support(s)/induce(s) sporulation, in addition to vegetative growth of the fungi. Above all, large requirements of Trichoderma spp. based BCAs (50% of fungal BCAs [4]) and their subsequent demands for enormous amounts of wastes will serve two purposes: (1)

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Fig. 3. Schematic comparison of conidia requirements in conventional and alternative (non-conventional) routes of Trichoderma production.

sustainable Trichoderma spp. based BCAs production and (2) waste management in a novel and environment-friendly way (reduced green house gases via incineration; and reduced hazard during land spreading-Trichoderma treated).

aged and emphasized, thereby, facilitating standardization and commercialization of Trichoderma spp. based BCA. In fact, bioassay methods need to be made more simpler, quantitative and standardized for better comparisons.

5.5. Standard bioassay for quality control

5.6. Trichoderma spp. based formulations

Last but not the least, similar to other biopesticides, standard bioassay technique for Trichoderma spp. is mandatory. However, this arena of Trichoderma spp. based BCAs is perhaps least considered by researchers. Bioassay protocols simulating in situ environment under in vitro conditions by best possible means (diet incorporation technique, droplet test) for Trichoderma, as standard is still awaited. Meanwhile, literature is replete with either, rudimentary protocols, such as, measurement of inhibition zone and viable spore count [15,67,80–82,140], lytic enzyme activity/metabolites [10,11] or, cumbersome greenhouse/field study [82,141]. Consequently, there are several examples where in vitro high potential Trichoderma spp. failed under in situ. Fortunately, there are occasional reports on development of simpler bioassay techniques for Trichoderma spp., which could simulate precise in situ conditions, such as, microdilution method based on infinite inhibition concentration [142–144]; and tubular bioassay system to measure production of antagonistic metabolites at the antagonist–pathogen interface [83]. Furthermore, for initial screening of potential Trichoderma spp. for biocontrol, a reliable, image analysis based quantitative evaluation bioassay of in vitro antagonism was also reported [145]. However, further bioassay research needs to be encour-

For obvious reasons, formulation development of BCAs is one of the most important steps in the overall production process. Ideally, formulation ensures protection of the active ingredients (spores, conidia, mycelial germlings of antagonistic fungi) from extreme pHs, lower humidity, chemicals and UV radiation. Formulated BCAs exhibit antagonistic action without being affected by the adverse environmental factors. Many researchers have reported different types of formulations of Trichoderma, e.g., invert emulsion [146]; cane molasses amendment [147]; seed coating [148]; pregelatinized starch-flour granules [149]; wettable powder [64]; alginate pellets [108]; gluten matrix [150]; and application techniques, e.g., seed treatments [151,152]; soil amendments [153]; honey bee [154]; bumble bee [154]; composted chicken manure [16]; composted cow manure [155]. Lewis et al. [156] reported that T. hamatum and T. virens were effective in disease prevention (>80%) and pathogen reduction (>75%) under greenhouse studies when amended with bran flakes or alginate pellets. However, in general, conidia without any amendments were ineffective. Furthermore, Bae and Knudsen [108] and McLean et al. [157] have demonstrated that when introduced in soil, the biocontrol activity of Trichoderma spp. was formulation dependent.

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Table 5 Comparison between Bacteria and Trichoderma spp. based BCAs Bacteria

Trichoderma sp.

Mode of action on pests through ingestion [136] Mass production normally by liquid fermentation [136] They have been used extensively in field [187] Many have been reported as plant growth enhancers, e.g., Rhizobium [201] Limited use in bioremediation of recalcitrants [202]

Usually, mode of action on pests/weeds through contact [104] Mass production normally by solid-state fermentation [118] Limited reported uses and market exploitation [4] Promote general plant growth and nutrition too [104] Widely exploited in bioremediation methods of recalcitrants [28]

5.7. Integrated pest management with Trichoderma spp. Many researchers have compared the efficiency of Trichoderma based BCAs with conventional chemical fungicides [64,152,155,158,159]. In comparison to chemical fungicide—azoxystrobin, the biocontrol showed by T. virens was as low as 33.3% disease infestation with 0.3% severity according to Fisher’s LSD test (P = 0.05) [64]. Using factorial design for in vitro experiment and randomized block design for field experiment, integration of T. harzianum (105 spores/ml/g seed) and carboxin (2 g/kg seed) for seed treatment resulted in enhanced seed germination (12.0–14.0%) and grain yields (42.6–72.9%) and reduced wilt incidence (44.1–60.3%) [158]. Similarly, other researchers have reported that the biocontrol exhibited by many Trichoderma spp. was greater than chemical fungicides. Thus, it could be concluded that Trichoderma based BCAs were competitive to their conventional chemical counterparts. Meanwhile, combination of Trichoderma based BCAs and chemical fungicides (integrated control) have been reported to be much more effective than either of the two alone. In fact, integrated control by means of Trichoderma and chemical pesticides combinations is becoming popular owing to cost economics as well as sustainable approach [152,158,159]. Furthermore, use of Trichoderma in integrated pest management makes them potential partners in pest control for quick and safer measures. 6. Market potential Presently, Trichoderma spp. based products are considered as relatively novel type of BCAs. In comparison to B. thuringiensis (Bt) biopesticides, their market size is quite small (Bt shares about 97% of overall biopesticides), they fall in remaining 3% bracket, which also comprises viral and nematode based biopesticides. The current status of Trichoderma spp. based BCAs is unlikely, especially, if we consider the positive features of Trichoderma spp. over bacteria as depicted in Table 5. In addition, the actual/true market size is vague and only scattered information could be obtained based on registered as well as non-registered biofungicides (Fig. 2). However, a general consent is that Trichoderma spp. based BCAs share about 60% of all fungal based BCAs and an increasing number of Trichoderma spp. based BCAs products are registered regularly. Moreover, field application/trials throughout the world is being accepted and many biopesticide companies are endorsing these products on regular basis (Table 3). The innate qualities (e.g., simulta-

neous biocontrol and growth promotion) of Trichoderma spp. based BCAs are driving factors for their steadily cumulating success. 7. Conclusions Trichoderma spp. play major role as biocontrol agents, owing to their capabilities of ameliorating crop-yields by multiple role, such as biopesticide, bioherbicides and plant growth promotion. Information on the classification of the genus, Trichoderma, mechanisms of antagonism and role in plant growth promotion has been well documented. However, fast paced current research in this field should be carefully updated for the fool-proof commercialization of the fungi. In order to enhance marketability of these fungi as BCAs, feasible commercial production processes are of utmost importance. Pursuit for cheaper and alternative substrates and optimal operating parameters to increase conidia production is on, and several encouraging results are being reported by researchers worldwide. Thus, it is expected that in near future, exploitation of these interesting BCAs would be maximized. Acknowledgements The authors are sincerely thankful to Natural Sciences and Engineering Research Council of Canada (Grants A4984, STP235071, Canada Research Chair) for financial support. The views and opinions expressed in this article are those of authors. Last but not the least, the authors would also like to thank Dr. Guillemond Ouellette (Histology, Host-pathogen Interaction—Natural Resources Canada) for his valuable suggestions to this work. References [1] C. Vincent, G. Hallman, B. Panneton, F. Fleurat-Lessard, Management of agricultural insects with physical control methods, Annu. Rev. Entomol. 48 (2003) 261–281. [2] G.E. Templeton, D.K. Heiny, Improvement of fungi to enhance mycoherbicide potential, in: J.M. Whipps, R.D. Lumsden (Eds.), Biotechnology of Fungi for Improving Plant Growth, Cambridge University Press, UK, 1989, pp. 127–151 (Chapter 6). [3] I. Chet, Biotechnology in Plant Disease Control, John Wiley and Sons, New York, 1993. [4] J.M. Whipps, R.D. Lumsden, Commercial use of fungi as plant disease biological control agents: status and prospects, in: T. Butt, C. Jackson, N. Magan (Eds.), Fungal Biocontrol Agents: Progress, Problems and Potential, CABI Publishing, Wallingford, 2001, pp. 9–22.

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[28]

[29] [30] [31]

[32]

[33]

[34]

[35]

[36] [37]

[38]

[39] [40]

[41] [42]

[43]

[44]

[45]

[46]

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