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Background Little is known about intestinal fungi in IBS patients whose gut bacteria have been investigated a lot. In order to explore causal relationship between IBS and gut mycobiome, and use gut fungi to diagnose or even treat IBS, further characterization of it in IBS is required. Methods Fifty-five diarrhea-predominant IBS (D-IBS) patients fulfilling Rome III criteria, and 16 healthy controls (HC) were recruited. Fresh fecal samples were collected and used for 16s rRNA and ITS2 high-throughput sequencing. Diversity and composition of gut bacteria and fungi, as well as bacterial-fungal interactions in D-IBS patients, were characterized. Specific fungal taxa differentiating D-IBS from HC were recognized by LEfSe and RandomForest methods, and their association with clinical symptoms was assessed by Spearman's correlation. Results Diarrhea-predominant irritable bowel syndrome patients showed abnormal (IBS-dysbiosis) or normal (HC-like IBS) fecal bacterial structure and diversity compared with healthy controls. However, fecal fungal signatures differed absolutely between D-IBS and HC, which indicated a more susceptible alteration of gut fungi than bacteria in D-IBS. Fecal fungi showed significant correlations with IBS symptoms, especially Mycosphaerella, Aspergillus, Sporidiobolus, and Pandora which were identified to potentially differentiate D-IBS from HC. Moreover, compared with HC there were markedly declined bacterial-fungal interactions in D-IBS, in which Candida changed from negative to positive correlations with bacteria, and Eurotium changed from positive correlations to irrelevance, while Debaryomyces gained negative correlations with bacteria. Conclusions Gut fungal dysbiosis and altered bacterial-fungal interactions were present in patients with D-IBS, and gut fungi could be used to diagnose D-IBS.

The Dothideomycete Pseudocercospora fijiensis, previously Mycosphaerella fijiensis, is the causal agent of black Sigatoka, one of the most destructive diseases of bananas and plantains. Disease management depends on fungicide applications, with a major contribution from sterol demethylation-inhibitors (DMIs). The continued use of DMIs places considerable selection pressure on natural P. fijiensis populations, enabling the selection of novel genotypes with reduced sensitivity. The hitherto explanatory mechanism for this reduced sensitivity was the presence of non-synonymous point mutations in the target gene Pfcyp51, encoding the sterol 14α-demethylase enzyme. Here, we demonstrate a second mechanism involved in DMI sensitivity of P. fijiensis. We identified a 19-bp element in the wild-type (wt) Pfcyp51 promoter that concatenates in strains with reduced DMI sensitivity. A polymerase chain reaction (PCR) assay identified up to six Pfcyp51 promoter repeats in four field populations of P. fijiensis in Costa Rica. We used transformation experiments to swap the wt promoter of a sensitive field isolate with a promoter from a strain with reduced DMI sensitivity that comprised multiple insertions. Comparative in vivo phenotyping showed a functional and proportional up-regulation of Pfcyp51, which consequently decreased DMI sensitivity. Our data demonstrate that point mutations in the Pfcyp51 coding domain, as well as promoter inserts, contribute to the reduced DMI sensitivity of P. fijiensis. These results provide new insights into the importance of the appropriate use of DMIs and the need for the discovery of new molecules for black Sigatoka management.

Heliconia rostrata (false bird of paradise, Heliconiaceae) is native to South America but is an important herbaceous perennial ornamental plant in India and globally. In December 2016, a foliar blight was observed on six ten-month-old H.…

Phenylphenalenones, polycyclic aromatic natural products from some monocotyledonous plants, are known as phytoalexins in banana (Musa spp.). In this study, (1) H nuclear magnetic resonance (NMR)-based metabolomics along with liquid chromatography and mass spectrometry were used to explore the chemical responses of the susceptible 'Williams' and the resistant 'Khai Thong Ruang' Musa varieties to the ascomycete fungus Mycosphaerella fijiensis, the agent of the black leaf Sigatoka disease. Principal component analysis discriminated strongly between infected and non-infected plant tissue, mainly because of specialized metabolism induced in response to the fungus. Phenylphenalenones are among the major induced compounds, and the resistance level of the plants was correlated with the progress of the disease. However, a virulent strain of M. fijiensis was able to overcome plant resistance by converting phenylphenalenones to sulfate conjugates. Here, we report the first metabolic detoxification of fungitoxic phenylphenalenones to evade the chemical defence of Musa plants.

BACKGROUND Evaluation of 4-phenylphenalenones and structural analogues against the fungal pathogen Mycosphaerella fijiensis (causal agent of black sigatoka disease in bananas) under light-controlled conditions uncovered some key structural features for the design of photodynamic compounds. RESULTS Structure–activity relationship analysis revealed the importance of a chromophoric aryl-ketone and a steroidomimetic structural motif in the activity of the assayed compounds. The results pointed to 1,2-dihydro-3H-naphtho[2′,1′:3,4]cyclohepta[1,2-b]furan-3-one, which displayed an activity in the range of propiconazole but with photodynamic behaviour. CONCLUSION The present work demonstrates that 1,2-dihydro-3H-naphtho[2′,1′:3,4]cyclohepta[1,2-b]heterocyclic-3-one derivatives can be used as potential lead compounds for the development of fungicides, relying on a dual mode of action. © 2015 Society of Chemical Industry

Diplodia pinea (causing diplodia blight), Fusarium circinatum (causing pitch canker), and Mycosphaerella spp. (causing needle blight) are examples of major fungal agents that damage Pinus spp. These diseases have a major negative impact on commercial plantation production, in addition to the risk of being introduced to native forest ecosystems. This study aimed to model the spatial risk of major forest diseases to Monterey pine plantations in northern Spain, to provide a basis for more focused management strategies. Generalized linear mixed-effects models were used to identify empirical relationships between environmental variables and disease occurrence. Field surveys and laboratory tests confirmed that D. pinea caused shoot blight in 24% of sampled plantations. Fusarium circinatum and Mycosphaerella spp. were detected in 15 and 71% of analysed plantations, respectively. The results confirmed that disease risk was strongly related to summer precipitation, moderately related to solar radiation, followed by summer temperature and tree age. The predicted probability of disease incidence was >20, 98 and 85% for Mycosphaerella spp., F. circinatum and D. pinea, respectively. The resultant maps from this study may be used to identify high-risk areas for increased monitoring and awareness of fungal diseases.

The Dothideomycetes represents a large and diverse array of fungi in which prominent plant pathogens are over-represented. Species within the Cochliobolus, Alternaria, Pyrenophora and Mycosphaerella (amongst others) all cause diseases that threaten food security in many parts of the world. Significant progress has been made over the past decade in understanding how some of these pathogens cause disease at a molecular level. It is reasonable to suggest that much of this progress can be attributed to the increased availability of genome sequences. However, together with revealing mechanisms of pathogenicity, these genome sequences have also highlighted the capacity of the Dothideomycetes to produce an extensive array of secondary metabolites, far greater than originally thought. Indeed, it is now clear that we appear to have only scratched the surface to date in terms of the identification of secondary metabolites produced by these fungi. In the first half of this review, we examine the current status of secondary metabolite research in the Dothideomycetes and highlight the diversity of the molecules discovered thus far, in terms of both structure and biological activity. In the second part of this review, we survey the emerging techniques and technologies that will be required to shed light on the vast array of secondary metabolite potential that is encoded within these genomes. Experimental design, analytical chemistry and synthetic biology are all discussed in the context of how they will contribute to this field.

Pseudocercospora fijiensis, the causal agent of black Sigatoka disease of banana (Musa spp.) (BLSD), is considered to be the major economic threat for banana cultivated for export (de Lapeyre de Bellaire et al., 2010). The disease has a …

Previously, we have determined the nonhost-mediated recognition of the MfAvr4 and MfEcp2 effector proteins from the banana pathogen Mycosphaerella fijiensis in tomato, by the cognate Cf-4 and Cf-Ecp2 resistance proteins, respectively. These two resistance proteins could thus mediate resistance against M. fijiensis if genetically transformed into banana (Musa spp.). However, disease resistance controlled by single dominant genes can be overcome by mutated effector alleles, whose products are not recognized by the cognate resistance proteins. Here, we surveyed the allelic variation within the MfAvr4, MfEcp2, MfEcp2-2 and MfEcp2-3 effector genes of M. fijiensis in a global population of the pathogen, and assayed its impact on recognition by the tomato Cf-4 and Cf-Ecp2 resistance proteins, respectively. We identified a large number of polymorphisms that could reflect a co-evolutionary arms race between host and pathogen. The analysis of nucleotide substitution patterns suggests that both positive selection and intragenic recombination have shaped the evolution of M. fijiensis effectors. Clear differences in allelic diversity were observed between strains originating from South-East Asia relative to strains from other banana-producing continents, consistent with the hypothesis that M. fijiensis originated in the Asian-Pacific region. Furthermore, transient co-expression of the MfAvr4 effector alleles and the tomato Cf-4 resistance gene, as well as of MfEcp2, MfEcp2-2 and MfEcp2-3 and the putative Cf-Ecp2 resistance gene, indicated that effector alleles able to overcome these resistance genes are already present in natural populations of the pathogen, thus questioning the durability of resistance that can be provided by these genes in the field.

BACKGROUND: A key challenge for designing RNAi-based crop protection strategies is the identification of effective target genesinthepathogenicorganism.Inthisstudy,invitroantifungalactivitiesofasetofsyntheticdouble-strandedRNAmolecules on spore germination of two major pathogenic fungi of banana, Fusarium oxysporum Schlecht f. sp. cubense WC Snyder & HN Hans (Foc) and Mycosphaerellafijiensis Morelet (Mf) were evaluated. RESULTS: All the tested synthetic dsRNAs successfully triggered the silencing of target genes and displayed varying degrees of potential to inhibit spore germination of both tested banana pathogens. When Foc dsRNAs were applied to Foc spores, inhibition ranged from 79.8 to 93.0%, and from 19.9 to 57.8% when Foc dsRNAs were applied to Mf spores. However, when Mf dsRNAs were applied on Mf spores, inhibition ranged from 34.4 to 72.3%, and from 89.7 to 95.9% when Mf dsRNAs were applied to Foc spores. CONCLUSION: The dsRNAs for adenylate cyclase, DNA polymerase alpha subunit and DNA polymerase delta subunit showed high levels of spore germination inhibition during both self- and cross-species tests, making them the most promising targets for RNA-mediated resistance in banana against these fungal pathogens. c � 2013 Society of Chemical Industry

Mycosphaerella species that cause the ‘Sigatoka disease complex’ account for significant yield losses in banana and plantain worldwide. Disease surveys were conducted in the humid forest (HF) and derived savanna (DS) agroecological zones from 2004 to 2006 to determine the distribution of the disease and variation among Mycosphaerella species in Nigeria. Disease prevalence and severity were higher in the HF than in the DS zone, but significant (P

Since 1911, dothistroma needle blight, caused by Dothistroma septosporum, has been recorded in most European countries. In the Czech Republic, the fungus has become an important disease of pines since 2000, especially Austrian pines in plantations of Christmas and ornamental trees. The aim of this study was to analyse the population structure, gene flow and mode of reproduction of this pathogen. Microsatellite and mating-type markers were analysed in a Dothistroma population in the southeastern part of the country using reference isolates from other European countries. The haplotypic diversity was high, with 87 unique and 13 shared haplotypes (probable clones) identified in 121 samples. Based on structure analysis, the isolates were divided into two populations, with an uneven distribution over the sampling sites. The grouping of the sites to the populations did not follow a geographical pattern because certain isolates that were sympatrically co-occurring at the same site were placed in different populations. Tests for random mating (the index of association and a parsimony tree-length permutation test) showed a significant clonal mode of reproduction in most cases, but the intrapopulation haplotypic diversity is unexpectedly high. Although a teleomorphic stage of D. septosporum has not been previously observed in the Czech Republic, the high intrapopulation haplotypic diversity can be explained by infrequent sexual reproduction consistent with the occurrence of both mating types.

Reconstructing and characterizing introduction routes is a key step towards understanding the ecological and evolutionary factors underlying successful invasions and disease emergence. Here, we aimed to decipher scenarios of introduction and stochastic demographic events associated with the global spread of an emerging disease of bananas caused by the destructive fungal pathogen Mycosphaerella fijiensis. We analysed the worldwide population structure of this fungus using 21 microsatellites and 8 sequence-based markers on 735 individuals from 37 countries. Our analyses designated South-East Asia as the source of the global invasion and supported the location of the centre of origin of M. fijiensis within this area. We confirmed the occurrence of bottlenecks upon introduction into other continents followed by widespread founder events within continents. Furthermore, this study suggested contrasting introduction scenarios of the pathogen between the African and American continents. While potential signatures of admixture resulting from multiple introductions were detected in America, all the African samples examined seem to descend from a single successful founder event. In combination with historical information, our study reveals an original and unprecedented global scenario of invasion for this recently emerging disease caused by a wind-dispersed pathogen.

SUMMARY Background: Banana (Musa spp.) is grown throughout the tropical and subtropical regions of the world.The fruits are a key staple food in many developing countries and a source of income for subsistence farmers. Bananas are also a major, multibilliondollar export commodity for consumption primarily in developed countries, where few banana cultivars are grown. The fungal pathogen Mycosphaerella fijiensis causes black leaf streak disease (BLSD; aka black Sigatoka leaf spot) on the majority of edible banana cultivars grown worldwide.The fact that most of these cultivars are sterile and unsuitable for the breeding of resistant lines necessitates the extensive use of fungicides as the primary means of disease control. BLSD is a significant threat to the food security of resource-poor populations who cannot afford fungicides, and increases the environmental and health hazards where large-acreage monocultures of banana (Cavendish subgroup, AAA genome) are grown for export. Taxonomy: Mycosphaerella fijiensis M. Morelet is a sexual, heterothallic fungus having Pseudocercospora fijiensis (M. Morelet) Deighton as the anamorph stage. It is a haploid, hemibiotrophic ascomycete within the class Dothideomycetes, order Capnodiales and family Mycosphaerellaceae. Its taxonomic placement is based on DNA phylogeny, morphological analyses and cultural characteristics. Disease symptoms and host range:Mycosphaerella fijiensis is a leaf pathogen that causes reddish-brown streaks running parallel to the leaf veins, which aggregate to form larger, darkbrown to black compound streaks.These streaks eventually form fusiform or elliptical lesions that coalesce, form a water-soaked border with a yellow halo and,eventually,merge to cause extensive leaf necrosis. The disease does not kill the plants immediately, but weakens them by decreasing the photosynthetic capacity of leaves, causing a reduction in the quantity and quality of fruit, and inducing the premature ripening of fruit harvested from infected plants. Although Musa spp. are the primary hosts of M. fijiensis, the ornamental plant Heliconia psittacorum has been reported as an alternative host. New opportunities: Several valuable tools and resources have been developed to overcome some of the challenges of studying this host‐pathogen system. These include a DNAmediated fungal transformation system and the ability to conduct targeted gene disruptions, reliable quantitative plant bioassays,diagnostic probes to detect and differentiate M. fijiensis from related pathogens and to distinguish strains of different mating types, and a genome sequence that has revealed a wealth of gene sequences and molecular markers to be utilized in functional and population biology studies.

Dispersal processes of fungal plant pathogens can be inferred from analysis of spatial genetic structures resulting from recent range expansion. The relative importance of longdistance dispersal (LDD) events vs. gradual dispersal in shaping population structures depends on the geographical scale considered. The fungus Mycosphaerella fijiensis, pathogenic on banana, is an example of a recent worldwide epidemic. Founder effects in this species were detected at both global and continental scale, suggesting stochastic spread of the disease through LDD events. In this study, we analysed the structure of M. fijiensis populations in two recently (_1979-1980) colonized areas in Costa Rica and Cameroon. Isolates collected in 10-15 sites distributed along a _250- to 300- km-long transect in each country were analysed using 19 microsatellite markers. We detected lowto- moderate genetic differentiation among populations in both countries and isolation by distance in Cameroon. Combined with historical data, these observations suggest continuous range expansion at the scale of banana-production area through gradual dispersal of spores. However, both countries displayed specific additional signatures of colonization: a sharp discontinuity in gene frequencies was observed along the Cameroon transect, while the Costa Rican populations seemed not yet to have reached genetic equilibrium. These differences in the genetic characteristics of M. fijiensis populations in two recently colonized areas are discussed in the light of historical data on disease spread and ecological data on landscape features. (Resume d'auteur)

Summary Mycosphaerella leaf diseases represent one of the most important impediments to Eucalyptus plantation forestry. Yet they have been afforded little attention in Uruguay where these trees are an important resource for a growing pulp industry. The objective of this study was to identify species of Mycosphaerellaceae and Teratosphaeriaceae resulting from surveys in all major Eucalyptus growing areas of the country. Species identification was based on morphological characteristics and DNA sequence comparisons for the Internal Transcribed Spacer (ITS) region of the rDNA operon. A total of ten Mycosphaerellaceae and Teratosphaeriaceae were found associated with leaf spots and stem cankers on Eucalyptus. Of these, Mycosphaerella aurantia, M. heimii, M. lateralis, M. scytalidii, Pseudocercospora norchiensis, Teratosphaeria ohnowa and T. pluritubularis are newly recorded in Uruguay. This is also the first report of M. aurantia occurring outside of Australia, and the first record of P. norchiensis and T. pluritubularis in South America. New hosts were identified for Kirramyces gauchensis, M. aurantia, M. marksii, M. lateralis, M. scytalidii, P. norchiensis, T. molleriana, T. ohnowa and T. pluritubularis. Interestingly K. gauchensis, which has been known only as a stem pathogen, was isolated from leaf spots on E. maidenii and E. tereticornis. The large number of Mycosphaerellaceae and Teratosphaeriaceae occurring in Uruguay is disturbing and raises concerns regarding the introduction of new pathogens that could threaten not only Eucalyptus plantations but also native forests.

Summary The most abundant metabolite of the eucalyptus leaf spot pathogen Mycosphaerella cryptica was extracted from agar cultures. The structure of the compound was elucidated by detailed studies of NMR and MS data and by comparison with derivatives. The compound is a previously undescribed diphenylether structurally related to pannaric acid found in lichens. Culture extracts from another species of Mycosphaerella isolated from the same environment yielded 5-hydroxymethylfuran3-carboxylic acid, a furan acidic compound previously isolated from a basidiomycete fungus. Assays for bioactivity of these metabolites revealed no evidence for antimicrobial activity. Some phytotoxicity was seen on newly emerged leaves of Eucalyptus globulus, but not on juvenile or adult leaves, when treated with either metabolite.

A collection of Mycosphaerella fijiensis, in total 71 isolates from 11 countries, of which 54 isolates were derived from the Ugandan Lake Victoria region, were analysed with regard to molecular markers and benomyl resistance to characterize the population structure. Forty-seven alleles from seven restriction fragment length polymorphism (PCR-RFLP) and five microsatellite markers were scored and utilized in the study. Using analysis of molecular variance (AMOVA), no significant genetic differentiation could be found between isolates from different leaves compared to isolates from a single leaf (P = 0.07). Exact tests of the allelic frequencies revealed no differences between isolates from different districts or between single-leaf-derived isolates compared to isolates derived from different leaves (P > 0.05 in all cases except two, which, however, were shown to be of type-I error). Resistance to benomyl, analysed both by PCR and fungicide screening, revealed that all isolates were susceptible. Compiled information from the various datasets implied that the Ugandan M. fijiensis population in the Lake Victoria Basin constitutes a homogenous population, probably as the result of a recent founder event.

Ribosomal coding DNA was sequenced and compared in 95 isolates of Mycosphaerella spp. collected in Nigeria and single nucleotide polymorphism (SNP) was used to identify the species and to determine the genetic structure of the sampled geographical populations. Using reference GenBank accessions with intercontinental distributions as controls, and shared species-specific SNPs in these control accessions, 84 (88·4%) isolates that grouped into 14 SNP haplotypes were identified as M. fijiensis, while 11 (11·6%) isolates represented by seven SNP haplotypes were characterized as M. eumusae. None of the isolates were either M. musicola or M. musae. The presence of M. fijiensis and M. eumusae in the collection was further confirmed using previously published species-specific probes designed on actin and β-tubulin gene sequences. A pairwise comparison of the population genetic distances revealed significant genetic differentiation between most populations (P

BACKGROUND:Mycosphaerella fijiensis Morelet causes black sigatoka, the most important disease in bananas and plantains. Disease control is mainly through the application of systemic fungicides, including sterol demethylation inhibitors (DMIs). Their intensive use has favoured the appearance of resistant strains. However, no studies have been published on the possible resistance mechanisms. RESULTS: In this work, the CYP51 gene was isolated and sequenced in 11 M. fijiensis strains that had shown different degrees of in vitro sensitivity to propiconazole, one of the most widely used DMI fungicides. Six mutations that could be related to the loss in sensitivity to this fungicide were found: Y136F, A313G, Y461D, Y463D, Y463H and Y463N. The mutations were analysed using a homology model of the protein that was constructed from the crystallographic structure of Mycobacterium tuberculosis (Zoff.) Lehmann & Neumann. Additionally, gene expression was determined in 13 M. fijiensis strains through quantitative analysis of products obtained by RT-PCR. CONCLUSION: Several changes in the sequence of the gene encoding sterol 14α-demethylase were found that have been described in other fungi as being correlated with resistance to azole fungicides. No correlation was found between gene expression and propiconazole resistance. Copyright © 2009 Society of Chemical Industry

In a disease complex, different pathogen species cause similar symptoms on a common host plant species. These pathogens commonly develop simultaneously in a field and can then infect the same host individuals. Co-occurring pathogens may affect each other, through antagonism and/or synergism. An important question is the ecological and pathological consequences of co-occurrence of pathogenic species within a disease complex. By using two pathogens (Mycosphaerella pinodes and Phoma medicaginis var. pinodella) of the Ascochyta blight complex, this study aimed at describing how co-occurrence affects the development of pathogens and disease severity, and at defining the prerequisites for interactions between pathogens. Results showed that the presence of the two pathogens on the same host plant organ limited the disease development and their reproduction. Damages caused by the two pathogens, however, increased when plants that had been previously inoculated were inoculated with the other species. The development of one pathogen species can be affected by a subsequent pathogen inoculation. Co-occurrence of pathogens within a disease complex can then limit or favour plant damages and pathogens reproduction. It would be one of the main forces that shape pathogen community structures and therefore the dynamics of diseases in field.

International audience; To investigate the hypothesis that disrupting pathogen movement within the plant canopy could slow the development of aschochyta blight, the effect of pea canopy architecture on splash dispersal of Mycosphaerella pinodes-conidia was studied in controlled conditions using a rainfall simulator generating rain events (2 mm) in still air. In intra-plant dispersal experiments, a source constituted by a semi-leafless pea plant with a single infectious lesion (108 pycnidia per cm2 of lesion, 1685 conidia per pycnidium) was placed in the middle of eight healthy target plants. Spore deposition was estimated by the number of lesions that developed on each stipule of the source (auto-deposition) and target (allo-deposition) plants after incubation. Rates of deposition on the source and target plants were 0·53 and 0·47, respectively. On the source plant, most of the spores were splashed downwards, with few spores remaining at the infectious node and very few spores moving upwards. In inter-plant dispersal experiments, potted plants were grouped to constitute 1-m2 canopies. A range of canopy architectures was achieved by using different plant densities and growth stages. A suspension of conidia was placed in the centre of each canopy. Resulting horizontal dispersal gradients were generally described by a negative exponential model. Canopies with a leaf area index (LAI) greater than 0·48 produced gradients with slopes that were not significantly different. A less dense canopy (LAI 0·36) produced a significantly steeper slope. Half-distances were short and ranged between 1·6 and 6·5 cm. The barrier rate, calculated as the ratio of the mean number of lesions assessed on isolated plants to the mean number of lesions assessed on plants in canopies, increased with increasing canopy LAI

Twelve microsatellite markers were developed for population analyses of the fungal pathogen, Dothistroma septosporum. Intersimple sequence repeat polymerase chain reaction (ISSR-PCR) and an enrichment protocol (fast isolation by amplified fragment length polymorphism of sequences containing repeats [FIASCO]) were both used to identify 28 unique microsatellite regions in the genome. From 22 primer pairs designed, 12 were polymorphic. These markers, screened on two populations representing 42 isolates, produced 40 alleles across all loci with an allelic diversity of 0.09-0.76 per locus. Cross-species amplification showed variable success with Dothistroma rhabdoclinis and Mycosphaerella dearnessi and some sequence variation within isolates of Dothistroma pini. These markers will be used to further study the population structure and diversity of D. septosporum.

BACKGROUND: In order to understand the bioactivity of Myoporum bontioides A. Gray against plant pathogens and determine its active ingredients, the inhibitory activities of methanol extracts from M. bontioides against Fusarium oxysporum f. sp. niveum (E. F. Smith) Snyder & Hansen, Pestalotia mangiferae P. Henn., Thielaviopsis paradoxa (De Seynes) v. Hohnel, Colletotrichum musae (Berk. & M. A. Curtis) v. Arx, Alternaria alternata (Fr.) Keissler, Mycosphaerella sentina (Fr.) Schroter and Sphaceloma fawcettii Jenk. were evaluated using a growth rate method, and the active ingredient was isolated by activity-directed isolation and identified by determination and analysis of IR, 1H NMR, 13C NMR and mass spectra and correlative physical constants. RESULTS: The results showed that the extracts from stems and leaves of M. bontioides exhibited inhibitory activity against the seven fungi, with > 58% inhibition at 10 g L−1 after 72 h. The active compound was isolated and identified as (−)-epingaione, and showed inhibitory activity against the above seven fungi. The inhibitory activity against P. mangiferae was the highest, with an EC50 value of 77 mg L−1. The EC50 values against the other six fungi were 147–245 mg L−1. (−)-Epingaione also inhibited spore germination of F. oxysporum f. sp. niveum, T. paradoxa and S. fawcettii. CONCLUSION: (−)-Epingaione demonstrated broad-spectrum inhibitory activity against plant pathogenic fungi and is promising for exploitation as a fungicide. Copyright © 2007 Society of Chemical Industry

The responses of juvenile leaves of two Eucalyptus species, with contrasting susceptibility to infection by Mycosphaerella leaf disease, were compared. The anatomical changes, accumulation of phenolics, suberin, lignin and anthocyanin and the retention of chlorophyll were studied in leaf lesions of varying developmental stages caused by species of Mycosphaerella. Enhanced resistance of Eucalyptus nitens in southern Australia was attributed to the formation of an effective lignified and suberized necrophylactic periderm, to restrict pathogen spread. Leaves of E. nitens contained isobilateral palisade which resulted in both abaxial and adaxial cell division and the initiation of a strong reinforced cellular zone from an early lesion stage. Eucalyptus globulus formed a slower, distorted necrophylactic periderm through hypertrophic changes to existing mesophyll and limited cell divisions of the single adaxial palisade layer. Deposits of lignin and suberin did not occur until later in lesion development, which were not effective in preventing further disease development. From this study it is hypothesized that tolerance of eucalypts to Mycosphaerella pathogens may be associated with constitutive mesophyll density.

Black Sigatoka caused by Mycosphaerella fijiensis Morelet is the most dangerous and devastating disease of banana around the world. Disease control is carried out by integrating cultural, genetic and chemical measures. Mycosphaerella musicola has been replaced by M. fijiensis wherever Black Sigatoka has been introduced in America and elsewhere, and remains as a significant problem at sites located at relatively high altitudes. To optimize fungicide applications for disease management a presymptomatic quantification of fungal proteins in leaves is desirable. In this study, we describe the generation of a mouse monoclonal antibody (Mab) reactive to a high-molecular weight antigens from M. fijiensis mycelial single ascospore in vitro culture, but not reactive to mycelial antigens from M. musicola, M. musae and M. minima single ascospore cultures. A rabbit-specific polyclonal antibody against the same M. fijiensis antigen, reactive also to fungal secreted proteins, was able to discriminate naturally M. fijiensis infected from healthy leaves as well as other concomitant fungi. The Mab was used as a capturing reagent and the polyclonal preparation as a second antibody for a triple antibody enzyme-linked immunosorbent assay able to quantify mycelial protein antigens in a range of 10–40 μg / ml and differentiate it from healthy banana leaf extracts. The aim of this study was the analytical setting up of a Mab-based immunoassay for the quantification of mycelial and secreted proteins of M. fijiensis. It is intended for the further establishment and optimization of an asymptomatic leaf assay for an improved forecast and warning system applied to Black Sigatoka disease management.

Resistance to Mycosphaerella pinodes in field pea (Pisum sativum L) is a quantitative trait, and expression of resistance is substantially influenced by environment. The disease reaction to mycosphaerella blight of four crosses (F 2:4 and F 2:5 populations) was assessed in field trials at Vegreville, AS, Canada. Broad-sense heritability of resistance was quite high (0.62-0.81). Narrow-sense (realized) heritability was moderate (0.43-0.57), indicating that additive genetic factors contributed substantially to the resistance phenotype. Significant improvement in resistance among the lines developed from selected individuals indicates that genetic improvement based on progeny testing would be effective. This study demonstrated that resistance to M. pinodes can be improved through progeny selection from crosses of the most resistant lines.

Mycosphaerella blight, caused mainly by Mycosphaerella pinodes (Berk. and Blox.) Vestergren, is the most important foliar disease on field pea (Pisum sativum L.) in western Canada. A quantitative trait analysis of resistance to M. pinodes was undertaken on five crosses with reciprocals of P. sativum to examine broad-sense (H 2 ) and narrow-sense (h 2 ) heritability, minimum number of genes involved (MNG), mid-parent heterosis (MPH), cytoplasmic inheritance, and epistasis. Mean H 2 was 0.75 (range 0.67-0.80) and mean h 2 was 0.59 (range 0.41-0.70), indicating that additive variance is important and that improvement in resistance can be achieved through breeding. Mean MNG was 2.16 genes (range 0.06-6.22), indicating that genes for resistance differed among parent lines. Mean MPH was 50% (range 47-57%), indicating that heterosis did not influence the expression of resistance to M. pinodes. There was no difference between the mean of any F 1 population and its reciprocal, indicating lack of maternal inheritance. The mean of the epistatic points was -0.01 (range -0.1 to 0.12), indicating that epistasis was not important in these crosses. These results will further the understanding of the natural genetic diversity for disease resistance to M. pinodes in P. sativum.

Summary Mycosphaerella leaf disease (MLD) can cause significant discoloration, necrosis and defoliation in commercial plantations of young Eucalyptus globulus. This study investigated the spectral reflectance changes that result in E. globulus foliage from Mycosphaerella infection. Spectral reflectance measurements were made on healthy and infected E. globulus foliage in the visible and near-infrared wavelengths (400–1000 nm). Reflectance (R) at 760 nm produced the largest difference between the extremes of disease severity classes. The wavelength most sensitive to infection severity was R678 nm. Other sensitive wavelengths were located between 480 and 500 nm. The wavelengths near 708 and 550 nm appeared relatively insensitive to infection severity. The reflectance index best correlated to leaf infection was R678/R550 (r = 0.841, p

Benzimidazoles are systemic fungicides widely used for the control of Black Sigatoka. They exert their antifungal activity by binding to β-tubulin and disrupting fungal cell division. The specific mode of action of benzimidazoles, along with their intensive use, has favoured the appearance of resistance in Mycosphaerella fijiensis, a situation which has precluded their use in several countries. Studies made in different fungi have identified several single nucleotide polymorphisms (SNP) associated with resistance to these fungicides. Despite the economic importance of this disease and the appearance of resistance to benzimidazoles, studies of the molecular mechanisms involved in resistance of this fungus have not been carried out. In this study, a fragment of the β-tubulin gene of 10 M. fijiensis isolates with three different levels of resistance to benomyl, was amplified and sequenced. The analysis of the sequences showed a change of adenine instead of cytosine in codon 198 of all the β-tubulin genes obtained from the isolates classified as having medium or high resistance. The single nucleotide change found, allowed the development of a molecular method based on polymerase chain reaction amplification and restriction digestion, which is able to differentiate resistant from susceptible isolates. The method was validated on a total of 44 isolates collected from different regions from Colombia, showing a 100% correlation between the presence of the resistant allele and the ability of the fungal isolate to grow in at least 10 μg a.i./ml benomyl. This method will be a valuable tool for monitoring benomyl resistance in M. fijiensis.

Summary Eucalyptus spp. are among the most widely planted exotic trees in Ethiopia. Several damaging leaf pathogens are known from Eucalyptus spp. worldwide. Of these, Mycosphaerella spp. are among the most important, causing the disease known as Mycosphaerella leaf disease (MLD). Characteristic symptoms of MLD include leaf spot, premature defoliation, shoot and twig dieback. Recent disease surveys conducted in Ethiopian Eucalyptus plantations have revealed disease symptoms similar to those caused by Mycosphaerella spp. These symptoms were restricted to E. globulus trees growing in several localities in south, south western and western Ethiopia. The aim of this study was to identify the fungi associated with this disease. This was achieved by examining ascospore germination patterns, anamorph associations and sequence data from the Internal Transcribed Spacer (ITS) region of the rRNA operon, for representative isolates. Several different ascospore germination patterns were observed, suggesting that more than one species of Mycosphaerella is responsible for MLD on E. globulus in Ethiopia. Analysis of sequence data showed that three Mycosphaerella spp., M. marksii, M. nubilosa and M. parva were present. This is the first report of these three species from Ethiopia and represents a valuable basis on which to build further studies in the region.

Summary The species composition of the endophytic mycobiota in leaves of Japanese beech trees (Fagus crenata) and the sources for leaf infections were studied in a forest reserve situated in central eastern Honshu, Japan. To clarify the mechanism of infection of leaves, half of the branches were covered with polyethylene bags and species composition and levels of endophytic fungal infection were then compared with those of unbagged controls. Isolations were carried out from the leaves, petioles, and current-year twigs of both, bagged and unbagged branches. Additionally, species composition was detected in overwintered terminal buds of beech trees and in the leaves of potted seedlings that had been placed in the field in different seasons. The species assemblage of the unbagged leaves, petioles, and current-year twigs was dominated by Mycosphaerella buna, Ascochyta fagi, Periconiella sp., and Tritirachium sp. Other frequently recovered species were Xylaria sp., Phomopsis sp., and Tubakia dryina. Mycosphaerella buna and A. fagi were never isolated from leaves on bagged branches. A. fagi was, however, detected on both bagged and unbagged petioles and current-year twigs at comparatively low isolation frequencies. The detection of Periconiella sp. on all occasions in both bagged and unbagged leaves was a characteristic feature that differs from those of the other three dominant endophytic fungi. The fungus was also detected without significant differences in bagged and unbagged petioles and current-year twigs on most sampling dates. Furthermore, Periconiella sp. was isolated from immature twigs inside the bud scales. Tritirachium sp. was frequently detected in unbagged leaves and petioles and in both bagged and unbagged current-year twigs, and rarely in bagged leaves and petioles, but was never recovered from terminal buds. The results of the potted seedling experiments revealed that all four dominant species had airborne inocula. The infection of leaves by M. buna occurs exclusively by airborne propagules, i.e. ascospores in spring and conidia in autumn. In Periconiella sp. hyphal growth of the fungus from immature twigs inside the buds into the leaf tissues was suggested in addition to infection by airborne inocula. Tritirachium sp. hyphae were suggested to grow from previous- to current-year twigs. Ascochyta fagi was present in the outermost scales of overwintered terminal buds, but no systemic growth of the fungus into the petioles and current-year twigs was observed. Our technique of covering the branches before new leaves unfolded was effective in preventing infection by airborne inocula of endophytic fungi. Resume La composition specifique et les voies d'infection des principaux champignons endophytes des feuilles de Hetre japonais (Fagus crenata) ont eteetudiees dans une reserve forestiere situee dans le Centre-Est de Honshu, au Japon. Afin d’elucider le mecanisme d'infection des feuilles, la composition specifique et le niveau d'infection des champignons endophytes ont ete compares entre un lot de branches ensachees dans des poches en polyethylene et un lot comparable de branches temoins non ensachees. Des isolements ont ete realises a partir des feuilles, petioles et rameaux de l'annee, sur les branches ensachees et non ensachees. De plus, la composition specifique a eteetudiee dans des bourgeons terminaux de Hetre a la fin de l'hiver et dans les feuilles de plantes en pots placees au champ a differentes periodes de l'annee. L'assemblage d'especes dans les feuilles, petioles et rameaux de l'annee des branches non ensachees est domine par Mycosphaerella buna, Ascochyta fagi, Periconiella sp., et Tritirachium sp. Xylaria sp., Phomopsis sp., et Tubakia dryina sont egalement frequemment obtenus. Mycosphaerella buna et A. fagi n'ont jamais ete isoles a partir de feuilles de branches ensachees. Ascochyta fagi a toutefois ete isole, a une frequence relativement faible, a partir de petioles et rameaux de l'annee, aussi bien sur branches ensachees que non ensachees. La detection systematique de Periconiella sp. a partir de feuilles ensachees ou non est un fait remarquable, qui differencie cette espece des 3 autres especes majeures d'endophytes. Le champignon a egalement ete isolea partir des petioles et rameaux de l'annee a la plupart des dates d’echantillonnage sans difference significative entre branches ensachees ou non. De plus, Periconiella sp. a ete isolea partir de rameaux immatures a l'interieur des ecailles de bourgeons. Tritirachium sp. a souvent ete detecte dans les feuilles et petioles non ensaches et dans les rameaux de l'annee ensaches ou non, mais rarement dans les feuilles et petioles ensaches et jamais dans les bourgeons terminaux. Les resultats de l'experimentation avec les plantes en pots ont montre que les quatre especes dominantes ont un inoculum aerien. L'infection des feuilles par Mycosphaerella buna se produit uniquement a partir de propagules a dissemination aerienne, ascospores au printemps et conidies en automne. Dans le cas de Periconiella sp., une colonization mycelienne des tissus foliaires a partir des rameaux immatures des bourgeons est suggeree en plus de l'infection par inoculum aerien. La croissance des hyphes de Tritirachium sp. depuis les rameaux de l'annee precedente vers les rameaux de l'annee pourrait se produire apres le debourrement. Ascochyta fagi est present dans les ecailles externes de bourgeons terminaux a la fin de l'hiver mais aucune croissance systemique du champignon n'a ete observee dans les petioles et rameaux de l'annee. La technique d'ensachage des branches avant debourrement s'est revelee efficace pour empecher l'infection des champignons endophytes par inoculum aerien. Zusammenfassung In einem Waldreservat im Zentrum des Ostens Honshus in Japan wurde die Artenzusammensetzung der endophytischen Mycobiota in Blattern der Japanischen Buche (Fagus crenata) untersucht. Die Halfte der untersuchten Aste wurde in Polyethylentuten eingepackt, um die Infektionswege dieser Pilze zu studieren. Blatter, Blattstiele und diesjahrige Astabschnitte von eingepackten Asten wurden mit solchen von nicht-eingepackten Asten (Kontrollen) hinsichtlich der Artenzusammensetzung und Haufigkeit der Pilzarten verglichen. In zusatzlichen Experimenten wurde die Artenzusammensetzung in uberwinterten Endknospen und in Blattern von Samlingen untersucht, die entweder von Juni bis Juli oder von September bis Oktober im Waldreservat exponiert wurden. Mycosphaerella buna, Ascochyta fagi, Periconiella sp. und Tritirachium sp. dominierten in Blattern, Blattstielen und diesjahrigen Astabschnitten der Kontrollen. Xylaria sp., Phomopsis sp. und Tubakia dryina waren weitere haufige Arten. Mycosphaerella buna und A. fagi konnten nie aus Blattern von eingepackten Asten isoliert werden. Ascochyta fagi konnte jedoch in geringer Haufigkeit in Blattstielen und diesjahrigen Astabschnitten von eingepackten und nicht-eingepackten Asten nachgewiesen werden. Periconiella sp. war an allen Probeterminen aus Blattern, Blattstielen und diesjahrigen Astabschnitten von eingepackten und nicht-eingepackten Asten isolierbar. Diese Art war aber nur in Blattern von nicht-eingepackten Asten signifikant haufiger. Periconiella sp. konnte auch aus Zweiginitialen in den Knospen isoliert werden. Tritirachium sp. wurde haufig in nicht-eingepackten Blattern, Blattstielen und diesjahrigen Astabschnitten sowie eingepackten Astabschnitten nachgewiesen. Dagegen war der Pilz in eingepackten Blattern und Blattstielen selten und in Knospen gar nicht vorhanden. Das Samlingsexperiment bestatigte, dass das Inokulum der vier dominanten Endophyten mit der Luft verbreitet wird. Die Infektion der Blatter durch Mycosphaerella buna erfolgt ausschliesslich durch luftverbreitete Sporen, im Fruhling durch Ascosporen und im Herbst durch Konidien. Fur Periconiella sp. wird vermutet, dass die Infektion der Blatter vorwiegend durch luftverbreitete Sporen besorgt wird. Zusatzliche Infektionen konnten von den endophytischen Thalli in den Zweiginitialen ausgehen. Das Myzel wurde dabei von den Zweiginitialen systemisch durch den Blattstiel ins frische Blatt hineinwachsen. Fur Tritirachium sp. wird angenommen, dass die Infektion diesjahriger Aste durch systemisches Myzelwachstum von den letztjahrigen Asten her erfolgt. Ascochyta fagi war in den aussersten Knospenschuppen von uberwinterten Endknospen vorhanden. Dennoch wurde kein systemisches Wachstum von den Knospenschuppen in die Blattstiele und in die diesjahrigen Zweige beobachtet. Die Vorbeugung luftburtiger Pilzinfektionen durch das Einpacken von Asten in Plastiksacke vor dem Blattaustrieb erwies sich als erfolgreich.

The worldwide destructive epidemic of the fungus Mycosphaerella fijiensis on banana started recently, spreading from South-East Asia. The founder effects detected in the global population structure of M. fijiensis reflected rare migration events among continents through movements of infected plant material. The main objective of this work was to infer gene flow and dispersal processes of M. fijiensis at the continental scale from population structure analysis in recently invaded regions. Samples of isolates were collected from banana plantations in 13 countries in Latin America and the Caribbean and in Africa. The isolates were analysed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and microsatellite molecular markers. The results indicate that a high level of genetic diversity was maintained at the plantation and the plant scales. The loci were at gametic equilibrium in most of the samples analysed, supporting the hypothesis of the existence of random-mating populations of M. fijiensis, even at the plant scale. A low level of gene diversity was observed in some populations from the Africa and Latin America-Caribbean regions. Nearly half the populations analysed showed a significant deviation from mutation-drift equilibrium with gene diversity excess. Finally, a high level of genetic differentiation was detected between populations from Africa (FST = 0.19) and from the Latin America-Caribbean region (FST = 0.30). These results show that founder effects accompanied the recent invasion of M. fijiensis in both regions, suggesting stochastic spread of the disease at the continental scale. This spread might be caused by either the limited dispersal of ascospores or by movements of infected plant material.

Single-copy restriction fragment length polymorphism (RFLP) markers were used to determine the genetic structure of #Mycosphaerella fijiensis#, the cause of black leaf streak (black Sigatoka) disease of banana and plantain, in the Torres Strait, Papua New Guinea (PNG), and the Pacific Islands. A moderate level of genetic variation was observed in all populations with genotypic diversity values of 60-78% of the theoretical maximum, and gene diversity (H) values between 0.269 and 0.336. All populations were at gametic equilibrium, and with the high level of genotypic diversity observed this indicated that sexual reproduction has a major role in the genetic structure of the #M. fijiensis# populations examined. Population differentiation was tested on several hierarchical scales. No evidence of population differentiation was observed between sites on Mer Island. A moderate level of population differentiation was observed within the Torres Strait, between Badu and Met Islands (Fst = 0.097). On a regional scale, the greatest differentiation was found between the populations of the Torres Strait and the Pacific. Populations from these regions were more closely related to the PNG population than to each other, suggesting they were founded in separate events from the same population.

Sodium dodecylbenzene sulphonate (SDBS) is an anionic synthetic detergent found in polluted sea aerosol and is known for its harmful effects on leaf surface ultrastructure on conifers and broadleaved trees. Four-year-old saplings of European beech were sprayed weekly for three consecutive growing seasons with either a 50 mg l−1 solution of SDBS in deionized water or with pure deionized water (control). Two- to three- year-old twigs were collected from SDBS-treated and control plants during the growing season one year after the last treatment to isolate endophytic fungi. The frequency of colonization by endophytic fungi was significantly lower on SDBS-treated plants (63.8%) than on control plants (85.4%). Multiple colonization of twigs occurred more frequently and diversity of endophyte species was higher in control plants than in SDBS-treated plants. Thirty-six fungal species were isolated from 360 twigs. Cladosporium cladosporioides, Coryneum compactum, Phialocephala dimorphospora, and a species each of Mycosphaerella and Phomopsis were the most abundant endophytes with frequencies of colonization of more than 5%. The abundance of the Phomopsis species proved to be significantly reduced by the SDBS treatment. Within the limits of the indoor experimental conditions, the obtained results suggest that long-term exposure of aerial parts of beech to SDBS can affect the amount and composition of endophytic fungal communities of lignified twigs. Degradation of the leaf epicuticular wax layer and changes of the assimilation capacity and leaf water content (transpiration) of the crowns are presumed to be responsible for the reduction of endophytic fungi detected in twigs of SDBS-treated plants.

Ascochyta blight of pea (Pisum sativum L.) is a fungal disease caused by Mycosphaerella pinodes (Berk. & Bloxham) Verstergren, Phoma medicaginis Malbr. & Roum. var. pinodella (L.K. Jones) Boerema, and Ascochyta pisi Lib. that can result in significant reductions to pea yield and quality. To characterize the genetics of resistance and to identify molecular markers for use in plant breeding, quantitative trait loci (QTLs) affecting Ascochyta blight resistance were mapped in F(2:3) and F(2:4) families produced from a cross between resistant breeding line 3148-A88 and susceptible cultivar Rovar. A linkage map containing 96 loci on 11 linkage groups was constructed for 133 families from this cross. Resistance of progeny lines to natural Ascochyta blight epidemics was examined in field trials at Medina, Western Australia, in 1997, 1998, and 1999. Disease severity was assessed on stems, leaves, and pods by means of separate rating scales. Because pea shows increased susceptibility to Ascochyta blight as it matures, plant reproductive stage was assessed at the time of disease scoring in the 1998 and 1999 trials. Thirteen QTLs were detected for Ascochyta blight resistance on seven linkage groups. Eight of these QTLs were detected in multiple environments or by multiple trait scores. One QTL for plant developmental stage was detected. Linkage of Ascochyta blight resistance QTLs to candidate genes including disease response genes and resistance gene analogs and of the QTL for plant reproductive stage to a pea homolog of the Arabidopsis thaliana CONSTANS gene controlling flowering time in response to photoperiod is discussed.

The present paper introduces improvements of the conventional selective amplification of microsatellite polymorphic loci (SAMPL) technique, that exploit AT-rich microsatellite primers. Generally, AT/AAT microsatellites are frequent components of eukaryotic genomes, but their ubiquity and polymorphic information content (PIC) could not be exploited yet, because standard SAMPL conditions did not allow amplifications. Here we report (i) on the design of new versatile AT-rich microsatellite primers, that are combined with (ii) a modified SAMPL adapter primer (called EcoRI-Short), and (iii) special polymerase chain reaction (PCR) amplification regimes. The novel SAMPL procedure expands the range of useful microsatellite primers to AT-rich sequences and produces a high number of bands and a clear banding pattern, and detects polymorphisms in otherwise nonpolymorphic genomes of plants (Dioscorea alata, D. rotundata) and a fungus (Mycosphaerella fijiensis).

Summary We previously reported on the in vitro antifungal activity of a crude whole plant extract from Eucomis autumnalis against seven economically important plant pathogenic fungi. A crude extract of the bulb showed similar in vitro mycelial growth inhibition of the same plant pathogenic fungi as well as that of an eighth fungus, Mycosphaerella pinodes, the cause of black spot or Ascochyta blight, in peas. Subsequently, fourth internode leaves were removed from 4 wk old pea plants, placed on moist filter paper in Petri dishes and inoculated with an M. pinodes spore suspension before and after treatment with the extract. The control of Ascochyta blight by different concentrations of the crude E. autumnalis extract was followed in vivo by leaf symptoms over a 6 day period at 20°C in a growth cabinet. The crude extract prevented M. pinodes spore infection of the leaves when the leaves were inoculated with spores both before or after treatment with the extract, confirming complete inhibition of spore germination. The crude E. autumnalis extract showed no phytotoxic reaction on the leaves even at the highest concentration applied.

Durum wheat ( Triticum durum ) is one of the most important cereal crops in Algeria with an annual production of approximately 1.3 million tonnes in 2015-2016. Septoria leaf blotch caused by Zymoseptoria tritici (syn. Mycosphaerella …

The genus Mycosphaerella Johanson contains many pathogens capable of causing a severe impact on the growth of susceptible eucalypt species. The lack of knowledge about which species are present in Tasmania and their potential risk to the plantation industry prompted this study into the Mycosphaerella species occurring on Eucalyptus globulus and Eucalyptus nitens plantations in Tasmania. A total of 36 plantation and ®ve road verge sites of E. globulus and E. nitens were sampled. Five Mycosphaerella species and three species from associated anamorph genera were isolated and identi®ed in Tasmania; Mycosphaerella nubilosa, Mycosphaerella cryptica, Mycosphaerella tasmaniensis, Mycosphaerella grandis, Mycosphaerella vespa, Coniothyrium ovatum, Sonderhenia eucalypticola and Sonderhenia eucalyptorum. The most frequently isolated species with the highest incidence and severity of infection were M. cryptica and M. nubilosa. These two species appear to have the greatest potential to damage juvenile eucalypt plantations in Tasmania. A link between Mycosphaerella vespa and Coniothyrium ovatum is described for the ®rst time.

Premise of the study: Large-scale population genetics studies are required to investigate the dispersal processes underlying the emergence of Mycosphaerella fi jiensis , a fungal pathogen of banana. To this end, we have developed an optimized genotyping procedure combining novel microsatellite markers and a modifi ed DNA extraction protocol. Methods and Results: Primers for tetranucleotide loci were designed directly from the recently published genome sequence of M. fijiensis. A total of 19 new polymorphic and easy-to-score markers were developed. Their use was combined with an adapted protocol for total DNA extraction starting from young lesions collected from banana leaves, thus avoiding a pathogen isolation step. Conclusions: The combination of the two technical developments presented here will permit the expansion of genotyping capacity in M. fijiensis, allowing large-scale analysis of samples from various geographic locations. (Resume d'auteur)

The host–pathogen interactions of Musa spp. and Mycosphaerella fijiensis were investigated in order to determine the function of secondary metabolites within the pathosystem of the Black Sigatoka disease. The pentaketide metabolites flaviolin, 2-hydroxyjuglone, juglone and 2,4,8-trihydroxytetralone (2,4,8-THT) of the pathogen were identified. The concentration of 2,4,8-THT was significantly increased by application of the synthetic compound tricyclazole and by natural activators extracted from the intercellular space of leaf tissue of resistant Musa cultivars. When inoculated host plants were treated with tricyclazole, extensive necrosis of both susceptible and resistant Musa cultivar leaves were observed. Plant defence mechanisms of resistant Musa cultivars were first detected as an activation of phenylalanine–ammonia lyase and the subsequent accumulation of post-infectional substances which blocked fungal growth. These results indicated the bivalent importance of 2,4,8-THT for host-specific reactions, depending on its concentration at different stages of pathogenesis. Early activation of fungal 2,4,8-THT metabolism by resistant Musa cultivars caused necrotic micro-lesions and elicitation of post-infectional defence reactions leading to incompatibility between pathogen and host plant; growth of the fungus on susceptible cultivars caused necrotizing doses of 2,4,8-THT only after the establishment of a compatible interaction and development of typical symptoms at late stages of pathogenesis. Zusammenfassung Die Wirt-Pathogeninteraktionen von Musa spp. und Mycosphaerella fijiensis wurden zur Bestimmung der Funktion sekundarer Metaboliten innerhalb des Pathosystems der ‘Schwarzen Sigatokakrankheit’ untersucht. Die Pentaketidmetaboliten Flaviolin, 2-Hydroxyjuglon, Juglon und 2,4,8-Trihydroxytetralon (2,4,8-THT) des Pathogens wurden identifiziert. Die Konzentration des 2,4,8-THT wurde durch die Anwendung des synthetischen Wirkstoffes Tricyclazol sowie durch naturliche Aktivatoren aus interzellularem Blattgewebe resistenter Musa-Sorten gesteigert. Die Inokulation Trizyclazol-behandelter Wirtspflanzen fuhrte zu ausgedehnten Nekrosen anfalliger und resistenter Musa-Sorten. Der erste erkennbare Abwehrmechanismus resistenter Musa-Sorten bestand in der Aktivierung von Phenylalanin-Ammoniumlyase und der Anreicherung von postinfektionellen Substanzen zur Hemmung des Pilzwachstums. Diese Ergebnisse belegten die bivalente Bedeutung des 2,4,8-THT fur wirtsspezifische Reaktionen, die von der Konzentration in verschiedenen Stadien der Pathogenese abhingen: Eine fruhe Aktivierung des pilzlichen 2,4,8-THT-Metabolismus bewirkte nekrotische Lasionen und die Elizitierung postinfektioneller Abwehrmechanismen, die zur Inkompatibilitat zwischen Pathogen und Wirtspflanze fuhrten; das Wachstum des Pilzes in anfalligen Sorten fuhrte erst nach Auspragung kompatibler Interaktionen zu nekrotisierenden Dosen von 2,4,8-THT und zur Entwicklung der typischen Symptome in spaten Phasen der Pathogenese.

SUMMARY Banana leaves showing different levels of black Sigatoka disease were collected from an unsprayed plantation in Costa Rica during two separate periods representing the wet to dry season transition (October 1993 – February 1994) and the dry to wet season transition (April – September 1995). Laboratory studies were used to investigate the relationship between the release of Mycosphaerella fijiensis ascospores and the amount of inoculum on banana plants showing different levels of infection, as assessed by leaf necrotic area. The number of perithecia present in the necrotic area was used as an indication of potential ascospore loads and was investigated as a series of regression equations. A series of rewetting and incubation regimes was used to investigate spore release under field conditions (21°C and 100% relative humidity in the early morning and 28°C, 60% relative humidity on days when it rained in mid-afternoon). Results suggest that rainfall, combined with a high temperature, may lead to peaks of ascospore release but without necessarily increasing overall numbers released over periods of up to 4 days and that a high level of spore release was less sensitive to changes in temperature once it had been initiated. The exact role of temperature in spore release is still unclear, however, as leaf samples kept at atypically low temperatures also released non-germinating ascospores. An average of 4.5 ascospores was released per perithecium. This does not resolve ambiguities in the literature regarding the number of ascospores present in each perithecium. A linear model relating the average ascospore numbers and necrotic area, using quick estimates of the amounts of necrotic area on the leaves of a random sample of plants across a plantation, is proposed, to give an indication of the relative amount of airborne inoculum potentially available between different plantations.

The characteristics of 50 isolates of Mycosphaerella pinodes and 17 isolates of Phoma medicaginis var. pinodella, originating from several regions of France where ascochyta blight is prevalent, were investigated using cultural, physiological, molecular and pathogenicity analyses. M. pinodes was distinguished from P. medicaginis var. pinodella on the basis of presence of pseudothecia, a higher proportion of larger, bicellular conidia, compared with the smaller, predominantly unicellular conidia of P. medicaginis var. pinodella, and a slower linear growth rate on agar under a 12-h light regime. RAPD analysis clearly distinguished the two species, which had low intraspecific variability. Although both species gave identical symptoms, they could be distinguished by their incubation period and aggressiveness, respectively, shorter and higher for M. pinodes. Virulence tests gave no definitive evidence for the existence of pathotypes among the M. pinodes isolates. Two unidentified isolates had similar characters to both M. pinodes and P. medicaginis var. pinodella in some features but were distinguished from them by their RAPD patterns.

The effect of interrupted wet periods on pycnidiospores of Mycosphaerella pinodes was studied by assessing spore viability, infection and disease development on pea seedlings. Pycnidiospores survived dry periods of up to 21 days after inoculation. Rewetting restored the infection capacity of the pycnidiospore, resulting in high levels of disease. The effects of wet–dry–wet cycles depended on when the dry period occurred during the infection process. No disease symptoms appeared when dry periods occurred during germination. A low level of disease occurred after rewetting in high relative humidity if the interruption of the wet period was long. However, a wet period resulting in leaf wetness after a dry period gave similar levels of infection to those achieved with a continuous wet period. Pycnidiospores formed appressoria but hyphae did not penetrate if a 6–12 h wet period preceded the dry period, and only a few flecks appeared during the dry period. Coalescent necrosis occurred when the dry period followed penetration. The disease was severe in each case when plants were returned to wet conditions after a period of dryness. Lesion development depended on the duration of the initial wet period, and the characteristics (temperature and duration) of both the dry period and the final wet period.

Fourteen polymorphic microsatellite markers were developed for Mycosphaerella fijiensis, a fungus causing the black sigatoka disease in banana. The sequenced genome of M. fijiensis was screened for sequences with single sequence repeats (SSRs) using a Perl script. Fourteen SSR loci, evaluated on 48 M. fijiensis isolates from Hawaii, were identified to be highly polymorphic. These markers revealed two to 19 alleles, with an average of 6.43 alleles per locus. The estimated gene diversity ranged from 0.091 to 0.930 across the 14 microsatellite loci. The SSR markers developed would be useful for population genetics studies of M. fijiensis.

The effects of Ascochyta blight (caused by Mycosphaerella pinodes) on the net photosynthetic rate of glasshouse-grown dried pea were studied on foliar discs and whole plants in pots showing different disease intensities, assessed visually using a disease scale (scores 0 to 5) or by an estimation of the fraction of leaf area with necrosis using an image analyser. The photosynthetic rate of foliar discs was measured using a leaf disc electrode; for plants it was assessed by CO2 exchange rate measurements in a closed chamber. A reduction in the net photosynthetic rate of diseased plants was correlated with an increase in disease score. A 53–56% reduction was found in foliar discs with a mean disease score of 3. On whole plants, mean scores of 3.5 and 4.0 were associated with reductions of 36% and 98% respectively. The disease also induced a decrease in the photosynthetic efficiency of the non-necrotic leaf area. The photosynthetic rate was zero when 30–40% of foliar area was necrotic on foliar discs and on whole plants. A function was derived from the foliar disc data that described the dependence of photosynthetic rate on the fraction of leaf area without necrosis. The model is discussed with reference to data obtained from entire plants and other pathosystems.