Multi-site fungicides suppress banana Panama disease, caused by Fusarium oxysporum f. sp. cubense Tropical Race 4.

Global banana production is currently challenged by Panama disease, caused by Fusarium oxysporum f.sp. cubense Tropical Race 4 (FocTR4). There are no effective fungicide-based strategies to control this soil-borne pathogen. This could be due to insensitivity of the pathogen to fungicides and/or soil application per se. Here, we test the effect of 12 single-site and 9 multi-site fungicides against FocTR4 and Foc Race1 (FocR1) in quantitative colony growth, and cell survival assays in purified FocTR4 macroconidia, microconidia and chlamydospores. We demonstrate that these FocTR4 morphotypes all cause Panama disease in bananas. These experiments reveal innate resistance of FocTR4 to all single-site fungicides, with neither azoles, nor succinate dehydrogenase inhibitors (SDHIs), strobilurins or benzimidazoles killing these spore forms. We show in fungicide-treated hyphae that this innate resistance occurs in a subpopulation of "persister" cells and is not genetically inherited. FocTR4 persisters respond to 3 µg ml-1 azoles or 1000 µg ml-1 strobilurins or SDHIs by strong up-regulation of genes encoding target enzymes (up to 660-fold), genes for putative efflux pumps and transporters (up to 230-fold) and xenobiotic detoxification enzymes (up to 200-fold). Comparison of gene expression in FocTR4 and Zymoseptoria tritici, grown under identical conditions, reveals that this response is only observed in FocTR4. In contrast, FocTR4 shows little innate resistance to most multi-site fungicides. However, quantitative virulence assays, in soil-grown bananas, reveals that only captan (20 µg ml-1) and all lipophilic cations (200 µg ml-1) suppress Panama disease effectively. These fungicides could help protect bananas from future yield losses by FocTR4.

Measurement of virulence in Zymoseptoria tritici through low inoculum-density assays.

Hitherto, pathogenicity assays with mutants or wildtype variants of Zymoseptoria tritici have been based on pycnidial counts, following inoculation of host leaves with high density inoculum. Here, we present data which suggest that high inoculum densities may mask deficiencies in virulence due to symptom saturation. We describe a low inoculum-density method which obviates this problem. This method can also be used to (i) interrogate the process of lesion formation in Z. tritici (ii) determine whether individuals of the same or different genotypes co-operate or compete during the establishment of apoplastic infections (iii) dissect the determinants of virulence, by assessing a given strain's stomatal penetration efficiency (SPE), its ability to spread within the apoplast and its pycnidiation efficiency. Such methodology can thus be used to investigate the reasons underpinning attenuated virulence in mutant or avirulent wildtype strains.

A novel role for catalase B in the maintenance of fungal cell-wall integrity during host invasion in the rice blast fungus Magnaporthe grisea.

Asexual spores of the rice blast fungus germinate to produce a specialized and melanized infection structure, the appressorium, which is pivotal to successful plant penetration. To investigate whether Magnaporthe grisea counteracts the toxic burst of H2O2 localized beneath the site of attempted invasion, we examined the temporal expression of five candidate antioxidant genes. Of these, the putatively secreted large subunit catalase CATB gene was 600-fold up-regulated in vivo, coincident with penetration, and moderately up-regulated in vitro, in response to exogenous H2O2. Targeted gene replacement of CATB led to compromised pathogen fitness; the catB mutant displayed paler pigmentation and accelerated hyphal growth but lower biomass, poorer sporulation, fragile conidia and appressoria, and impaired melanization. The catB mutant was severely less pathogenic than Guy 11 on barley and rice, and its infectivity was further reduced on exposure to H2O2. The wild-type phenotype was restored by the reintroduction of CATB into the catB mutant We found no evidence to support a role for CATB in detoxification of the host-derived H2O2 at the site of penetration. Instead, we demonstrated that CATB plays a part in strengthening the fungal wall, a role of particular importance during forceful entry into the host.

A role for the asexual spores in infection of Fraxinus excelsior by the ash-dieback fungus Hymenoscyphus fraxineus.

The invasive pathogen, ash dieback fungus Hymenoscyphus fraxineus, is spreading rapidly across Europe. It shows high levels of outcrossing and limited population structure, even at the epidemic front. The anamorphic (asexual) form produces prolific conidia, thought to function solely as spermatia (male gametes), facilitating gene flow between sympatric strains. Here, we show that conidia are capable of germination on ash leaves and in vitro, and can infect seedlings via leaves or soil. In leaves, germlings form structures resembling fruiting bodies. Additionally, H. fraxineus colonises ash debris and grows in soil in the absence of ash tissues. We propose an amended life-cycle in which wind-dispersed, insect-vectored or water-spread conidia infect ash and may sporulate in planta, as well as in forest debris. This amplifies inoculum levels of different strains in ash stands. In combination with their function as spermatia, conidia thus act to maximise gene flow between sympatric strains, including those originally present at low inoculum. Such mixing increases evolutionary potential, as well as enhancing the likelihood of gene introgression from closely-related strains or assimilation of further genetic diversity from parental Asian populations. This scenario increases the adaptability of H. fraxineus to new climates and, indeed, onto new host species.

Active diffusion and microtubule-based transport oppose myosin forces to position organelles in cells.

Even distribution of peroxisomes (POs) and lipid droplets (LDs) is critical to their role in lipid and reactive oxygen species homeostasis. How even distribution is achieved remains elusive, but diffusive motion and directed motility may play a role. Here we show that in the fungus Ustilago maydis ∼95% of POs and LDs undergo diffusive motions. These movements require ATP and involve bidirectional early endosome motility, indicating that microtubule-associated membrane trafficking enhances diffusion of organelles. When early endosome transport is abolished, POs and LDs drift slowly towards the growing cell end. This pole-ward drift is facilitated by anterograde delivery of secretory cargo to the cell tip by myosin-5. Modelling reveals that microtubule-based directed transport and active diffusion support distribution, mobility and mixing of POs. In mammalian COS-7 cells, microtubules and F-actin also counteract each other to distribute POs. This highlights the importance of opposing cytoskeletal forces in organelle positioning in eukaryotes.

Blumeria graminis secretes an extracellular catalase during infection of barley: potential role in suppression of host defence.

SUMMARY The obligate biotrophic fungal pathogen of barley, Blumeria graminis f.sp. hordei (Bgh), elicits a burst of H(2)O(2) in its host barley at sites of germ tube invasion. To evaluate whether this specialized pathogen has any antioxidant response to this oxidative burst, the Bgh catB gene was characterized and transcript-profiled together with other genes implicated in the management of oxidative stress (catalase-peroxidase, cpx; glutathione peroxidase, gpx; superoxide dismutase, sod1) and in comparison with the constitutively expressed Bghbeta-tubulin and elongation factor1 (ef1) genes. Gel-based and real-time RT-PCR revealed enhanced numbers of catB transcripts at mature primary germ tube and appressorium germ tube (AGT) stages in a susceptible host. Moreover, an anti-CATB polyclonal antibody, from Aspergillus fumigatus, which recognizes both native and recombinant Bgh CATB, revealed an intense circle of immunofluorescence at the host-pathogen interface at the AGT tip and within the halo area surrounding the host papilla. A new diaminobenzidine-based 'scavenger' assay revealed areas of H(2)O(2) clearing at sites of fungal invasion, provoking speculation that Bgh catalase activity may contribute to pathogenicity in Bgh.