Microsatellite markers for the fungal banana pathogens Mycosphaerella fijiensis, Mycosphaerella musicola and Mycosphaerella eumusae

We developed a total of 50 microsatellite markers for the three fungal pathogens causing the most important leaf spot diseases of banana: 32 loci for Mycosphaerella fijiensis are presented, and nine loci each for Mycosphaerella musicola and Mycosphaerella eumusae. All these loci were polymorphic within each species on a sample of isolates collected from various locations around the world. Within M. fijiensis and M. musicola, most of the loci tested (> 80%) in a sample of isolates from a single location in Cameroon were also polymorphic. Multiplex polymerase chain reaction systems were developed with 15 loci for M. fijiensis.     

Evaluation of plant extracts as an antagonist to mycelial growth of Mycosphaerella fijiensis morelet

Three plant extracts (rice husk, wood and bamboo) at different concentration were evaluated in vitro as an antagonist to mycelial growth of Mycosphaerella fijienesis on different culture media using spread plate and mycelia dry weight method. The plant extracts had significant effects on the mycelial growth of Mycosphaerella fijiensis. Rice husk extract at concentration of 1, 1.5, 2.5 and 5% completely inhibited the mycelia growth of M. fijiensis in malt extract broth (MEB) and at 2.5 and 5% on malt extract agar (MEA). Wood extract at concentration of 1 and 1.5% inhibited the mycelial growth of M. fijiensis and completely at concentration of 2.5 and 5% on MEA. Although complete inhibition was only observed at 5% concentration on MEA for bamboo extract, the evaluated plant extracts could be recommended for the control of M. fijiensis on a large-scale farming.     

A new mechanism for reduced sensitivity to demethylation‐inhibitor fungicides in the fungal banana black Sigatoka pathogen Pseudocercospora fijiensis

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.     

Reactive Oxygen Species and Cellular Interactions Between Mycosphaerella fijiensis and Banana

Globally, the banana plant (Musa spp) is the fourth most important crop after rice, wheat and corn (based on production in tons). It is cultivated in more than 100 tropical and subtropical countries, mainly by small producers and is a fundamental food source for millions of people. Black leaf streak disease (BLSD), caused by Mycosphaerella fijiensis Morelet (sexual phase) or Paracercospora fijiensis (Morelet) Deighton (asexual phase), is the main disease affecting the world??s banana culture. This disease has a wide geographical distribution accounting for losses exceeding 50% of global banana production. We conducted a comparative histocytological study on the kinetics of the infection process using three banana genotypes with phenotypes that differ in resistance to BLSD: Grand Naine (Susceptible), Pisang Madu (Partially Resistant) and Calcutta 4 (Resistant). Experiments were conducted under controlled conditions with the objective of characterizing the cellular interaction processes between M. fijiensis and Musa acuminata. Conidia germination occurred 24 hours after inoculation. Germination rates were high (97%) and there were no significant differences between the three genotypes (P?>?0.147). The Peroxidase enzyme and H₂O₂ were associated with a hypersensitivity-like reaction in the resistant genotype Calcutta 4, indicating a possible role of the enzyme or its product as defense mechanisms against M. fijiensis in banana plants.     

Microsatellite markers for the fungal banana pathogen Mycosphaerella musicola

Yellow Sigatoka caused by the ascomycete Mycosphaerella musicola Leach, is one of the most severe banana diseases worldwide, which spread in most banana growing areas, until Black Sigatoka, a more aggressive disease caused by Mycosphaerella fijiensis, appeared. Because of the highly devastating nature of the latter pathogen, recent research almost exclusively focused on M. fijiensis. To close the gap of knowledge and to study the population structure of M. musicola in Yellow Sigatoka‐infested areas, we cloned and characterized a versatile set of 26 polymorphic locus‐specific microsatellite markers.     

Selection ofMycosphaerella fijiensis-resistant cell lines from micro-cross sections of banana and plantain

Anin vitro selection system using microcross sections of banana and plantain cultivars belonging to AAA and AAB genomic groups were used to produce plants resistant against the Black Sigatoka disease. The fungus resistant plantlets were obtained in a double selection system. This involved in a first step the use of a fungal crude filtrate and in the second step the purified host-specific toxin 2,4,8-trihydroxytetralone extracted from the fungusMycosphaerella fijiensis (M. fijiensis), the causal agent of Black Sigatoka disease. Resistant plantlets obtained from the double selection system were inoculated with conidia ofM. fijiensis in a growth chamber to reproduce Black Sigatoka symptoms. Compared to non-treated control plantlets, which were highly susceptible to the fungus, 10.7–19.3% toxin-resistant plantlets which arose from tissues that went through the double selection system were resistant againstM. fijiensis. This technique of using micro-cross sections for selection on fungal toxins seems to be amenable to differentMusa genotypes for the production of fungus-resistant plants.F. A. Schulz died 11. 3. 1995     

Potential use of Trichoderma-based bioproduct for black leaf streak disease (Mycosphaerella fijiensis) management in the field

Organic banana production demands alternatives for controlling black leaf streak disease caused by Mycosphaerella fijiensis Morelet. The foliar application of a Trichoderma-based bioproduct increased the plant growth and provides some degree of control by reduction of the disease severity in two growing cycles of the banana plants cv. ‘Williams’.     

Phenylphenalenones protect banana plants from infection by Mycosphaerella fijiensis and are deactivated by metabolic conversion

Phenylphenalenones, polycyclic aromatic natural products from some monocotyledonous plants, are known as phytoalexins in banana (Musa spp.). In this study, ¹H 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.     

Polymerase chain reaction–restriction fragment length polymorphism markers for the fungal banana pathogen Mycosphaerella fijiensis

Polymerase chain reaction–restriction fragment length polymorphism markers were developed to study populations of the fungal banana pathogen Mycosphaerella fijiensis. Twelve markers were defined, 11 in anonymous and single‐copy nuclear DNA sequences and one in the internal transcribed spacer and 5.8S rDNA sequence. The polymerase chain reaction products obtained with locus‐specific primer pairs were digested with restriction enzymes to reveal polymorphism. Between five and 12 markers were polymorph in M. fijiensis populations from different geographical origins (Papua New Guinea, the Philippines, Cameroon and Latin America). The mean of allele number and gene diversity (expected heterozygosity) per locus in the different geographical populations ranged between 1.4 and 2.7 and 0.17 and 0.45, respectively.     

Positive selection and intragenic recombination contribute to high allelic diversity in effector genes of Mycosphaerella fijiensis,causal agent of the black leaf streak disease of banana

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.     

In vitro evaluation of Colombian plant extracts against Black Sigatoka (Mycosphaerella fijiensis Morelet)

In this work, 90 dichloromethane and methanol extracts obtained from 45 plants collected at the Natural Reserve Bremen – La Popa (Colombia) and at the Natural Regional Park Ucumarí (NRPU, Colombia) belonging to five botanical families were evaluated at 1000 mg/l, for their in vitro fungicide activity through the ascospore germ tube elongation and the measurement of the mycelial radial growth of Mycosphaerella fijiensis assays. The methanol extracts from the species Lycianthes acutifolia (Solanaceae) and Piper pesaresanum (Piperaceae); as well as, the dichloromethane extracts from P. pesaresanum and those from the Lauraceae family named Nectandra acutifolia and Ocoteca paulii, all inhibited M. fijiensis ascospore germination in 100% in the germinative tube elongation assay. With regards to the effects of the plant extracts on mycelial radial growth, the methanol extracts from P. pesaresanum and the dichloromethane one from N. acutifolia both showed 100% inhibition in this bioassay. Additionally, from the phytochemical screening on the dichloromethane and methanol extracts it was found that compounds such as alkaloids, phenols and terpenes were present in most of the extracts evaluated and they might be the cause of the antifungal activities reported.     

Use of the IRAP Marker to Study Genetic Variability in Pseudocercospora fijiensis Populations

Pseudocercospora fijiensis is the etiological agent of black Sigatoka, which is currently considered as one of the most destructive banana diseases in all locations where it occurs. It is estimated that a large portion of the P. fijiensis genome consists of transposable elements, which allows researchers to use transposon-based molecular markers in the analysis of genetic variability in populations of this pathogen. In this context, the inter-retrotransposon-amplified polymorphism (IRAP) was used to study the genetic variability in P. fijiensis populations from different hosts and different geographical origins in Brazil. A total of 22 loci were amplified and 77.3 % showed a polymorphism. Cluster analysis revealed two major groups in Brazil. The observed genetic diversity (H _E) was 0.22, and through molecular analysis of variance, it was determined that the greatest genetic variability occurs within populations. The discriminant analysis of principal components revealed no structuring related to the geographical origin of culture of the host. The IRAP-based marker system is a suitable tool for the study of genetic variability in P. fijiensis.     

Genetic discontinuities and disequilibria in recently established populations of the plant pathogenic fungus Mycosphaerella fijiensis

Dispersal processes of fungal plant pathogens can be inferred from analysis of spatial genetic structures resulting from recent range expansion. The relative importance of long‐distance 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 low‐to‐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.     

Contrasting introduction scenarios among continents in the worldwide invasion of the banana fungal pathogen Mycosphaerella fijiensis

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.     

Movement ofMycosphaerella fijiensis spores and sigatoka disease development on plantain close to an inoculum source

Investigations into the short-distance dispersal of ascospores and conidia ofMycosphaerella fijiensis and subsequent disease development, from point sources of inoculum, were carried out in a small plot of 100 initially uninfected plantain plants in Costa Rica during 1995. Plants were examined every 4 days from the time of planting in early May for the first appearance of disease symptoms. After 3 months, all plants were infected. Levels of inoculum within the plot were monitored with spore traps and weather data were collected. Results suggested that Black Sigatoka disease was spread on the wind, with conidia ofM. fijiensis probably responsible for short-distance dispersal and ascospores for spread of the disease over longer distances. No evidence was found to support splash dispersal of these spores.     

Musa methylated DNA sequences associated with tolerance toMycosphaerella fijiensis toxins

DNA methylation is an epigenetic phenomenon associated with gene silencing in transgenic plants, retrotransposons and virus infection. Expression analysis of specific genes in Arabidopsis methylation mutants showed an association between DNA methylation and gene expression. To determine whether DNA methylation is associated with resistance to black Sigatoka (BS) andMycosphaerella fijiensis (MF), we used anin vitro assay of mesophyll cell suspensions of reference cultivars with known resistance to BS. Methylation of CC^mGG sequences was evaluated by methylation-sensitive amplification polymorphism (MSAP) markers of reference cultivars and somaclonal variants to identify molecular markers associated with resistance to MF toxins and BS. Four MSAP markers were associated with resistance (MAR) to MF toxins. The MSAP markers show a high degree of sequence similarity with resistance gene analog and with retrotransposon sequences. The MSAP markers are useful as molecular indicators of tolerance to MF toxins and resistance to BS.     

Inheritance of black sigatoka disease resistance in plantain-banana (Musa spp.) hybrids

Black sigatoka (Mycosphaerella fijiensis Morelet), an airborne fungal leaf-spot disease, is a major constraint to plantain and banana (Musa spp.) production world-wide. Gaining further knowledge of the genetics of host-plant resistance will enhance the development of resistant cultivars, which is considered to be the most appropriate means to achieve stable production. Genetic analysis was conducted on 101 euploid (2x, 3x and 4x) progenies, obtained from crossing two susceptible triploid plantain cultivars with the resistant wild diploid banana Calcutta 4. Segregating progenies, and a susceptible reference plantain cultivar, were evaluated over 2 consecutive years. Three distinct levels of host response to black sigatoka were defined as follows: susceptible (< 8 leaves without spots), less susceptible (8–10) and partially resistant (> 10). Segregation ratios for resistance at the 2x level fitted a genetic model having one major recessive resistance allele (bs ₁) and two independent alleles with additive effects (bsr ₂ and bsr ₃). A similar model explains the results at the 4x level assuming that the favourable resistance alleles have a dosage effect when four copies of them are present in their respective loci (bs _i ⁴ ). The proposed model was further validated by segregation data of S ₁ progenies. Mechanisms of black sigatoka resistance are discussed in relation to the genetic model.     

Biologically Active Endophytic Streptomycetes from <Emphasis Type="Italic">Nothofagus</Emphasis> spp. and Other Plants in Patagonia

Endophytic streptomycetes have been isolated and characterized from several species of Nothofagus and other plants growing in the southern reaches of Patagonia. No endophytic streptomycete was obtained from any plant species studied in Northern Patagonia. However, from Southern Patagonia, biologically active Streptomyces spp. from several plant species were isolated. Each isolate, as studied by scanning electron microscopy (SEM), has small hyphae, some produce typical barrel-shaped spores in culture and each has some unique hyphal surface structures. Interestingly, although none has any detectable antibacterial killing properties, each has demonstrable killing activity against one or more pathogenic fungi including representative plant pathogenic organisms such as Phytophthora erythroseptica, Pythium ultimum, Sclerotinia sclerotiorum, Mycosphaerella fijiensis, and Rhizoctonia solani. The 16S rDNA sequences of the isolates were distinct from all other genetic accessions of Streptomyces in GenBank. However, isolate C-2 from Chiliotrichum diffusum (Compositae) is identical, in all respects, to isolate C-4 obtained from Misodendrum punctulatum (Loranthaceae). These results confirm that endophytic streptomycetes represent a novel source of biologically active microorganisms.     

Construction and characterization of a bacterial artificial chromosome library of the causal agent of Black Sigatoka fungal leaf spot disease of banana and plantain, <Emphasis Type="Italic">Mycosphaerella fijiensis</Emphasis>

A bacterial artificial chromosome library of the causal agent of the Black Sigatoka leaf spot disease of banana and plantain, Mycosphaerella fijiensis, has been constructed using a non-sphaeroplasting technique and characterized using both homologous and heterologous probes. After first and a second size selection of PFGE-fractionated DNA, a ligation was obtained using a 1:4 molar ratio (insert:vector). One hundred random clones were analyzed, and the mean insert size was estimated to be 90 kb. The range of the insert sizes was between 40 and 160 kb. The highest percentage of inserts belonged to the range between 80 and 100 kb; 32% of the inserts had 2 or 3 internal NotI sites. This library consists of 1920 clones, if the genomic size is at least 35 Mb, then this represents 4.9× genome equivalents, which was supported by hybridization results with homologous and heterologous probes. Blondy Canto-Canché and Diana Karina Guillén-Maldonado contributed equally to this work and should be regarded as co-first authors.