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Production of secondary metabolites
Endophytes produce toxic secondary metabolites to overcome host defence mechanisms when penetrating and colonizing their host plants. Schulz et al. (2002) termed such a relationship “balanced antagonism”. Endophytic secondary metabolites (alkaloids) were first recognised in grasses because of their detrimental effect to livestock. In 1977, Bacon et al. (1977) established that the endophytic fungus Epichlo typhina (Fr.) Tul produced alkaloids in tall fescue that were toxic to domestic mammals. Perennial rye grass infected with endophytes was later observed to cause a nervous disorder called “rye grass staggers” in cattle, sheep and deer (Gallagher et al., 1981, Rowan and Gaynor, 1986). It is now well known that toxicity in pasture grasses upon endophyte infection is associated with the ability of the endophyte to produce the alkaloid Lolitrem in the host (Kemp et al., 2007). Endophytes also influence the interaction of their host with other organisms by producing alkaloids that are toxic to herbivores (Siegel et al., 1990; Clement et al., 1997; Wilkinson et al., 2000; Bultman et al., 2004)
Four major classes of alkaloids are produced in the grass-endophyte symbiosis. These include pyrrolizidines (lolines), ergot alkaloids (ergovaline), indole diterpenes (lolitrem A, paxilline) and pyrrolopyzine (Bush et al., 1997). Lolines act as metabolic toxins and feeding deterrents to insects (Bush et al., 1997). They are not widely distributed among endophyte-infected grasses, but show the highest concentration in substantially infected grasses (Clay and Schardl, 2002). Lolines affect insect pests by causing reduction in growth rates or inhibiting reproduction (Siegel et al., 1990; Riedell et al., 1991). Ergovaline is toxic to invertebrate- and vertebrate herbivores such as cattle and has been isolated from tall fescue infected with N. coenophialum (Arechavaleta et al., 1992). Lolitrems occur in perennial rye grass infected with Neotyphodium endophytes. They have neurotoxic effect on sheep that feed on infected grass (Clay and Schardl, 2002). Pyrrolopyzine is represented by alkaloids such as peramine which is known to be toxic to insects such as the Argentine stem weevil (Listronotus bonariensis (Kuschel) (Rowan and Latch, 1994). The alkaloid is produced in planta by N. coenophialum, N. lolii and E. festucae in stems and leaves of tall fescue and rye grass.
Events leading to resistance induction
Rapid defence responses by a host depend on the ability of the host to recognize an invader or specific elicitor molecules (Garcia-Garrido and Ocampo, 2002). The latter may be secreted by the invader (exogenous elicitor) or may be components of the host cell wall released during penetration (endogenous elicitor) (Heath et al., 1996). Morphological recognition between an endophyte and its host starts when specific exudates are released by the host that initiate hyphal proliferation (Gianinazzi-Pearson et al., 1990). In mycorrhiza, a gus-derived diffusible signal molecule, called -glucuronidase fusion, is involved in attracting fungal hyphae to the host (Parniske, 2004). Recognition at the site of infection initiates cellular signalling processes that activate defence responses locally and systemically. Elicitor recognition is followed by several biochemical changes within the host. One of the earliest reactions is the change in plasma membrane permeability, subsequently leading to Ca2+ ion and proton influx (Ebel and Scheel, 1997). These ions are necessary for adequate induction of the oxidative burst, phytoalexin production and defence gene activation (Jabs et al., 1997; Pugin et al., 1997). Ca2+ signaling is very essential for the activation of defence responses in higher plants and Ca2+ ion influx is required for the activation of molecules involved in signal transduction, such as calmodulin and protein kinases (Heo et al., 1999).
Penetration of intact epidermal cells by fungal endophytes is a pre-requisite for the induction of resistance in host plants, as was demonstrated with the biocontrol non-pathogenic F. oxysporum-induced resistance in cucumber against the Fusarium wilt pathogen F. oxysporum f. sp. cucumerinum Owen (Qaher, 2006). Upon penetration of host cells, an oxidative burst (generation of reactive oxygen species (ROS)) results. It occurs in affected cells within seconds or minutes of plant cell wall contact with elicitor molecules (Bradley et al., 1992) and reaches maximum activity soon after induction. The oxidative burst is a common feature of all plant-fungal interactions (Grayer and Kokubun, 2001). ROS produced by plants include superoxides, hydrogen peroxide (H2O2) and singlet oxygen (Wojtaszek, 1997). ROS may have direct anti-microbial activity (Peng and Kuc, 1992; Mellersh et al., 2002; Vranova et al., 2002; Wang et al., 2008), act as messengers for activating defence responses (Chen et al., 1995; Jabs et al., 1997; Chamnongpol et al., 1998), may be involved in cell wall modification through POX-catalysed cell wall cross linking of protein polymers (Brisson et al., 1994), and may trigger the hypersensitive response (Levine et al., 1994). The hypersensitive response (cell death) during the oxidative burst results from a combined activity of ROS, sulphur and nitrogen intermediates which inactivate and destroy proteins, lipids and nucleic acids (Sedlarova et al., 2007). To counteract this effect, plants usually deploy enzymatic (POX and catalases) and non-enzymatic (phenolic compounds) antioxidants to avert damage to host cells (Vranová et al., 2002; Herbbete et al., 2003).
Isolation and identification of defence-related genes in EAHB
A total of 62 TDFs were successfully isolated from roots and rhizomes of endophyteinoculated EAHB plants. In this study, the word TDF is used to refer to fragments of the same length produced by the same primer combination. Of the 62 TDFs, 55 were successfully assigned putative identities (Table 2), while seven had no significant similarity to any protein in the non-redundant protein database in Genbank. TDFs were broadly classified into seven functional categories: defence/stress-related, primary metabolism, transport, signal transduction, cell wall biosynthesis, cell differentiation and development, and regulation (Fig. 1). Among TDFs with putative identities, those involved in cell differentiation and development, primary metabolism and defence/stress were most abundant, making 22, 21 and 15%, respectively, of the total number of TDFs. TDFs with similarities to proteins of unknown function made up 18% of total TDFs (Fig.1). Of the 62 TDFs, 67.7% were up-regulated in the cv Nabusa, 6.5% in cv Kayinja and 25.8% in both cultivars (Table 2). The majority of TDFs related to cell differentiation and development (75%), primary metabolism (75%) and defence/stress (62.5%) were up-regulated in the susceptible cv Nabusa. Only one TDF related to cell differentiation and development, and one related to primary metabolism, was up-regulated in the tolerant cv Kayinja. TDFs related to cell differentiation and development, primary metabolism and defence/stress up-regulated in both cultivars were 9.2, 9.2 and 37.5%, respectively. Among TDFs associated with cell differentiation and development, primary metabolism and defence/stress, only COI1 and a senescence protein were up-regulated in the rhizome of cv Nabusa.
Seven TDFs with similarity to defence-related genes, glycolate oxidase, COI1, cathepsin Blike protease, beta-N-acetyl hexosaminidase, two calmodulin-Ca2+ and LOX, were upregulated in the susceptible cv Nabusa (Table 2). Only three of these, COI1, calmodulin-Ca2+ and LOX, were also up-regulated in the tolerant cv Kayinja. TDFs with similarity to beta-Nacetyl hexosaminidase, calmodulin-Ca2+, COI1 and LOX were up-regulated 2 dai with isolates Emb2.4o and V5w2, while glycolate oxidase was up-regulated 2 dai with isolate Emb2.4o (Table 2). With the exception of glycolate oxidase, these TDFs remained up-regulated until 30 dai. Cathepsin B-like protease was up-regulated at 7 and 30 dai. Two TDFs with similarity to proteins involved in cell wall strengthening (cellulose synthase and 1,3-glucan synthase) were up-regulated in cv Nabusa alone (Table 2). 1,3-glucan synthase was up-regulated early (2 dai) and stayed up-regulated until 30 dai, while cellulose synthase was up-regulated at 7 dai and remained up-regulated until 30 dai. Differences were also observed between roots and rhizomes in TDF up-regulation. TDFs isolated from roots accounted for 86% of the total number of TDFs, compared to the 6 and 8% of TDFs obtained from the rhizome alone and from both the roots and rhizomes, respectively. Of the defence-related genes, only COI1 was up-regulated in both the roots and rhizomes of cv Nabusa, while none of the cell wallstrengthening genes was up-regulated in the rhizome.
CHAPTER 1 LITERATURE REVIEW: THE USE OF FUNGAL ENDOPHYTES, ESPECIALLY NON-PATHOGENIC FUSARIUM OXYSPORUM, IN PROTECTING BANANA PLANTS AGAINST PESTS
INTRODUCTION
BANANAS
PESTS AND DISEASES OF BANANA
FUNGAL ENDOPHYTES
THE INTERACTION BETWEEN BANANA AND NON-PATHOGENIC FUSARIUM OXYSPORUM ENDOPHYTES CONCLUSIONS
REFERENCES
CHAPTER 2 DEFENCE-RELATED GENE EXPRESSION IN SUSCEPTIBLE AND TOLERANT BANANAS (MUSA SPP.) FOLLOWING INOCULATION WITH NON-PATHOGENIC FUSARIUM OXYSPORUM ENDOPHYTE V5W2 AND CHALLENGE WITH RADOPHOLUS SIMILIS
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
CHAPTER 3 DIFFERENTIAL GENE EXPRESSION IN NEMATODE-SUSCEPTIBLE AND TOLERANT EAST AFRICAN HIGHLAND BANANAS (MUSA SPP.) FOLLOWING INOCULATION WITH NON-PATHOGENIC FUSARIUM OXYSPORUM ENDOPHYTES
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
DISCUSSION
REFERENCES
CHAPTER 4 ACTIVITIES OF PHENYLPROPANOID PATHWAY ENZYMES IN SUSCEPTIBLE AND TOLERANT BANANAS (MUSA SPP.) FOLLOWING INOCULATION WITH A NON-PATHOGENIC FUSARIUM OXYSPORUM ENDOPHYTE AND CHALLENGE WITH RADOPHOLUS SIMILIS
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
DISCUSSION
REFERENCES
CHAPTER 5 MARKING ENDOPHYTIC NON-PATHOGENIC FUSARIUM OXYSPORUM ISOLATES FOR CHEMICAL RESISTANCE AND WITH FLUORESCENT PROTEINS FOR USE IN PLANT COLONIZATION STUDIES
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
CHAPTER 6 DUAL INOCULATION OF FUSARIUM OXYSPORUM ENDOPHYTES: EFFECT ON PLANT COLONIZATION, PLANT GROWTH AND CONTROL OF RADOPHOLUS SIMILIS AND COSMOPOLITES SORDIDUS
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
