Indicadores Financieros

4.2.1

Arthroconidiation

Activation of the PcatA-EGFP reporter was also observed in E. festucae transformants growing through the agar overlay. Further analysis revealed a segmented appearance of the hyphae reminiscent of arthroconidia or arthrospores formed by a large variety of fungi in response to stress. Expression of the catA reporter revealed two important aspects of the arthroconidia morphology. Firstly, given the spore-specificity of catA expression in E. festucae, these experiments provided evidence that these structures are spore-like. Secondly, the strong reporter expression is indicative of oxidative stress and suggests H2O2-detoxification by catalase is a crucial

function in these arthroconidia.

There is a great diversity in the fungal species that form arthroconidia and while morphologically diverse, arthroconidia are generally recognised as asexual spore-like structures resulting from the segmentation of pre-existing hyphae (Barrera, 1986). Arthroconidia are an important means of transmission for many mammalian pathogenic fungi and consequently the literature is dominated by reports of medically important fungi such as Microsporum, Epidermophyton and Trichophyton dermatophytes as well as coccidioidomycosis-causing Coccidioides species (Farnoodian et al., 2009, Stevens, 1995). It has been proposed that these infectious propagules (arthroconidia)

+0* are important for the survival of the fungus in the hostile environment of the host and unsurprisingly certain environmental stresses induce arthroconidiation (Barrera, 1986). A variety of environmental factors including temperature, pH and O2 availability

stimulate arthroconidia production in a range of fungi and it is possible that one or more of these factors contributed to the observed arthroconidiation in E. festucae transformants (Yazdanparast & Barton, 2006).

Reduced oxygen tension beneath the agar overlay is thought to have a major contribution to formation of arthroconidia in E. festucae as reducing the O2 availability

to surface-grown cultures produced similar morphological effects. Furthermore, depletion of oxygen in these cultures also resulted in activation of the PcatA-EGFP reporter. This strongly suggests that the limited oxygen availability encountered by the protoplasts beneath the agar overlay is one of the key contributing factors towards arthroconidiation. While the morphology of microaerophilicallygrown cultures was similar to the originally observed arthroconidial form, they were not identical suggesting that while reduced O2 tension beneath the agar surface may contribute to

the formation of arthroconidia, other factors are also involved.

The finding that regenerating hyphae of M. oryzae also formed very similar structures when growing through the agar overlay is of great interest as it implies that this may be a more general phenomenon in fungi (A. Tanaka, unpublished data, 2012). If conversion to an arthroconidial state is required for growing through the agar overlay, these observations may have potential implications for isolating certain gene deletion mutants in E. festucae and, more broadly, a range of fungal species. From the morphological appearance of E. festucae cultures growing through the agar overlay, it is apparent that dramatic changes to the cellular structure take place. Hence, disruption of genes that confer cell wall defects may not be identified if mutants are incapable of growing through the agar overlay. Given the resemblance of arthroconidia to asexual spores, it is also possible that deletion of genes involved in sporulation may prevent strains defective in sporulation from penetrating the agar overlay. Furthermore, the strong PcatA-EGFP expression suggests oxidative stress is high in these cells, which may prevent oxidative stress-sensitive mutants growing through the overlay.

4.2.2

H

O

-sensitivity of spores

Wild-type E. festucae conidia (asexual spores) are markedly more sensitive than vegetative mycelia to H2O2. The elevated sensitivity of E. festucae spores to H2O2

+0+ examined the oxidative stress-sensitivity of 12 fungal species, including Humicola, Fusarium, Alternaria, Cladosporium, Penicillium and Aspergillus and found that spores of these strains could withstand much higher H2O2 concentrations than that tolerated by E. festucae in this study. The marked difference observed in the sensitivity of E. festucae spores may reflect the differences in lifestyles and ecological niches inhabited by these organisms. In many fungal-plant pathogen lifecycles, conidia play a central role in the disease cycle as the primary inoculum source for dissemination of the fungus. Conidia of B. cinerea are extremely resistant to stress such as H2O2 and often must survive

extended periods exposed to the environment before encountering a suitable host. In contrast, E. festucae is predominantly a mutualistic symbiont and is largely transmitted vertically through the seed. Therefore, it is not so strongly dependent on asexual conidia for its perpetuation of infection and may not have faced the same evolutionary selection pressures to produce spores tolerant to environmental stresses external to the plant, such as UV-induced oxidative stress.

4.2.3

Sporulation in planta

Isolation of asexual conidia from E. festucae can be challenging as it sporulates poorly in axenic culture. However, enhanced sporulation was observed 2-3 days after transferring mycelia onto the meristematic region of one-week old perennial ryegrass seedlings. Sporulation was observed on both slit-wounded and unwounded seedlings, indicating that the plant itself, rather than secondary effects related to a wounding response, induces sporulation. Molecules on the plant surface such as oxylipins have previously been shown to regulate sporulation of fungal pathogens Fusarium verticillioides and Colletotrichum graminicola on maize plants and similar molecules produced by L. perenne may induce sporulation in E. festucae (Gao et al., 2007).

The formation of epiphyllous hyphae and sporulation on the leaf surface has also been observed as part of the natural life cycle in later stages of the association (Moy et al., 2000). While infection naturally occurs through host florets by germinating ascospores during the sexual stage of the E. festucae lifecycle, the asexual sporulation observed on the epiphyllous mycelium may facilitate transmission to new hosts in the asexual phase of the fungal life cycle (Moy et al., 2000). Alternatively, environmental stresses such as nutrient starvation can induce asexual sporulation in fungi (Adams et al., 1998). It is possible that the sporulation observed in E. festucae cultures transferred to L. perenne seedlings reflects the poor nutrient availability on the plant surface. However, water agar alone did not induce extensive sporulation in E. festucae cultures. Furthermore, nutrient-dependent asexual sporulation in other fungi tends to occur in submerged cultures rather than surface-grown colonies (Adams et al., 1998). Thus, the

+0, plant surface appears to serve as an environmental cue to induce asexual sporulation.