Ascospore Germination Research Articles

Current understanding and future prospects for ash dieback disease with a focus on Britain

Abstract Hymenoscyphus fraxineus is an introduced ascomycete fungus which causes ash dieback and has resulted in widespread mortality of ash throughout Europe. Although H. fraxineus has been present on the continent for at least four decades, it was not identified until 2006. The first record of the pathogen in Britain came in 2012 although it was probably present a decade earlier. The most common host European ash (Fraxinus excelsior L.) is economically and ecologically important to Britain where the cost of ash dieback is estimated at billions of pounds. The impact of ash dieback has stimulated a major research response which we review with the aim of providing up-to-date information relevant to Britain and identifying knowledge gaps where research would contribute to improved disease mitigation. Hymenoscyphus fraxineus is an outcrossing fungus with high genotypic diversity; ascospores produced via sexual reproduction are critical to aerial dispersal and infection. Temperature, moisture, and ground cover influence pathogen fruit body development, the timing of ascospore release, and extent of ascospore germination; they also interact together to affect the likelihood of infection. In addition, stand characteristics, including tree density, tree height, and landscape fragmentation, affect disease dynamics with increased disease severity on moist sites with high ash density. Efforts at finding natural resistance in ash have identified genetic markers associated with disease tolerance, and gene expression analysis is providing insights into the basis of that resistance. Mainland European findings indicate that ash dieback makes trees more vulnerable to other pathogens, whilst endophytes in the ash phyllosphere can suppress infection by H. fraxineus. Possible tools for long-term control of ash dieback include (1) deployment of resistant ash, (2) quantitatively informed management practices based on microclimate models and better understanding of the pathogen life cycle, and (3) manipulation of biocontrol agents from the ash microbiome or pathogen mycoviruses.

Ascospore germination and comparative monocycle of neonectria fruit rot on apple and pear

Ascospore germination and comparative monocycle of neonectria fruit rot on apple and pear

Effects of Temperature on Spore Germination and Mycelial Growth of Calosphaeria pulchella, Cytospora sorbicola, and Eutypa lata Isolates Associated with Sweet Cherry Canker Diseases.

Although fungal canker diseases constitute a limiting factor to orchard productivity and longevity, little is known about the effects of temperature on spore germination and mycelial growth of the fungal causal agents. Accordingly, the germination of spores and colony growth of Calosphaeria pulchella, Cytospora sorbicola, and Eutypa lata were evaluated after incubation on 2% water agar and 4% potato dextrose agar, respectively, at 5, 10, 15, 20, 25, 30, 35, and 40°C. Temperature optima for spore germination and mycelial growth were derived from nonlinear models fitted to germination rates and colony diameter data. The optimal temperatures for spore germination of Cal. pulchella were 28.5°C for ascospores and 29.2°C for conidia. The optimal temperatures for Cyt. sorbicola conidia and E. lata ascospore germination were 25.8 and 23.1°C, respectively. The germination of ascospores and conidia of Cal. pulchella at temperatures below 15°C required an incubation time of at least 72 h. Ascospores of E. lata and conidia of Cyt. sorbicola germinated at 10°C after 36 h. The optimal temperature for colony growth of Cal. pulchella was 24.6°C, whereas it was 21.7°C for both Cyt. sorbicola and E. lata. Our study indicates that temperature requirements for basic biological functions are higher for Cal. pulchella than for Cyt. sorbicola and E. lata. The overall higher temperatures of California relative to other cherry-producing regions in the United States or worldwide could explain the prevalence of Calosphaeria canker in the state. Conversely, Cyt. sorbicola and E. lata appear better adapted to cooler temperatures.

Influence of Temperature and Wetness on Taphrina deformans Ascospore and Blastospore Germination: Disease Forecasting and Validation

Peach leaf curl is a fungal disease caused by Taphrina deformans, and it can severely affect the health and productivity of peach and nectarine trees (Prunus persica) if left unmanaged. This study was carried out to investigate the temperature and wetness conditions that affect the germination of blastospores and ascospores of local isolates of the fungus T. deformans. The results showed that the rate of both ascospore and blastospore germination was reduced as the temperature increased from 0 to 20 °C. A decrease in temperature from the range of 25 °C to 30 °C caused a reduction in the germination of conidia for both ascospores and blastospores. Ascospore and blastospore germination were totally inhibited at −3 and 35 °C. Under constant temperatures of 20 °C, the percentage of both ascospore and blastospore germination of T. deformans gradually increased as the wetness period increased from 9 to 15 h. However, there was no further increase in germination observed beyond the 15 h wetness period. Additionally, this study aimed to validate the predictive models of T. deformans, developed based on the favorable temperatures and leaf wetness durations, under the specific field conditions of Naoussa, Greece. The results indicate that while both the ascosporic and blastosporic models were capable of correctly predicting infection periods, there were differences in their predictions of infection risk. The ascosporic model predicted lower risk infection, which aligned well with the observed symptoms of the disease. In contrast, the blastosporic model predicted higher risk infection, but this did not match the actual intensity of the symptoms. Finally, this study also provided insights into the potential benefits of using predictive models to guide fungicide applications, potentially leading to more targeted and efficient disease management strategies for commercial peach orchards.

GUN Mutants: New Weapons To Unravel Ascospore Germination Regulation in the Model Fungus Podospora anserina.

In Podospora anserina as in many other Ascomycetes, ascospore germination is a regulated process that requires the breaking of dormancy. Despite its importance in survival and dispersal, ascospore germination in filamentous fungi has been poorly investigated, and little is known about its regulation and genetic control. We have designed a positive genetic screen that led to the isolation of mutants showing uncontrolled germination, the GUN (Germination UNcontrolled) mutants. Here, we report on the characterization of the gun1SG (Spontaneous Germination) mutant. We show that gun1SG is mutated in Pa_6_1340, the ortholog of Magnaporthe oryzae Pth2, which encodes a carnitine-acetyltransferase (CAT) involved in the shuttling of acetyl coenzyme A between peroxisomes and mitochondria and which is required for appressorium development. Bioinformatic analysis revealed that the mutated residue (I441) is highly conserved among Fungi and that the mutation has a deleterious impact on the protein function. We show that GUN1 is essential for ascospore germination and that the protein is localized both in mitochondria and in peroxisomes. Finally, epistasis studies allowed us to place GUN1 together with the PaMpk2 MAPK pathway upstream of the PaNox2/PaPls1 complex in the regulation of ascospore germination. In addition, we show that GUN1 plays a role in appressorium functioning. The pivotal role of GUN1, the ortholog of Pth2, in ascospore germination and in appressorium functioning reinforces the idea of a common genetic regulation governing both appressorium development and melanized ascospore germination. Furthermore, we characterize the second CAT encoded in P. anserina genome, Pa_3_7660/GUP1, and we show that the function of both CATs is conserved in P. anserina. IMPORTANCE The regulation of ascospore germination in filamentous fungi has been poorly investigated so far. To unravel new genes involved in this regulation pathway, we conducted a genetic screen in Podospora anserina, and we isolated 57 mutants affected in ascospore germination. Here, we describe the Germination UNcontrolled One (gun1SG) mutant, and we characterize the gene affected. GUN1 is a peroxisomal/mitochondrial carnitine-acetyltransferase required for acetyl coenzyme A shuttling between both organelles, and we show that GUN1 is a pleiotropic gene also involved in appressorium functioning similarly to its ortholog, the pathogenesis factor Pth2, in the plant pathogen Magnaporthe oryzae. Given the similarities in the regulation of appressorium development and ascospore germination, we speculate that discovering new genes controlling ascospore germination in P. anserina may lead to the discovery of new pathogenesis factors in pathogenic fungi. The characterization of GUN1, the ortholog of M. oryzae Pth2, represents a proof of concept.

Salt tolerance of Chaetomium globosum, a common indoor mold

Chaetomium globosum is in the Kingdom Fungi (Mycota), Phylum Ascomycota. Although C. globosum is a soilborne fungus, it also commonly infests water-damaged buildings. The hyphae and ascospores can be highly allergenic. C. globosum isolates found in buildings and homes were investigated to determine their halotolerance response to various salts. Malt extract agar (MEA) or broth was amended with either NaCl, KCl, CaCl2, or MgCl2 at concentrations from 0 to 2 M. Ascospore germination, ascospore synthesis, vegetative growth (hyphal), and perithecia development were measured. C. globosum ascospore germination was inhibited by 1 M NaCl-amended media, but not by 1 M KCl-amended media. Ascospore synthesis and vegetative growth were inhibited when grown in 1 M KCl, 1 M NaCl, 1 M MgCl2, and 1 M CaCl2-amended media. However, 100 mM CaCl2-amended plates inhibited ascospore synthesis. Growth of C. globosum in broth cultures showed similar growth patterns in salt-amended media. Perithecia synthesis was inhibited at 1 and 2 M concentrations of NaCl and KCl. Extracellular protein content was not different in the 0-100 mM treatments. However, extracellular protein synthesis as an indicator of vegetative growth and metabolic stress activity, was less in the 1 M NaCl-amended broth, and 7 to 10-fold lower in the 2 M NaCl-amended broth treatments. C. globosum isolates appeared to be halotolerant and only relatively high concentrations of salt inhibited the growth, ascospore synthesis, germination, and perithecia production. This research may have applications in controlling indoor mold growth and further the understanding of halotolerance in Chaetomium spp.

Comparing the Fungicide Sensitivity of Sclerotinia sclerotiorum Using Mycelial Growth and Ascospore Germination Assays.

The infection of the floral tissues of snap bean and other crops by Sclerotinia sclerotiorum, the causative agent of white mold, is by ascospores. Irrespective of the fungicide mode of action being evaluated, invitro fungicide sensitivity tests are conducted almost exclusively using mycelial growth assays. This is likely because of difficulties and time involved in sclerotial conditioning required to produce apothecia and ascospores. The objective of this research was to compare estimates of fungicide sensitivity between mycelial growth and ascospore germination assays for S. sclerotiorum. Sensitivity assays were conducted using serial doses of three fungicides commonly used to control white mold: boscalid, fluazinam, and thiophanate-methyl. A total of 27 isolates were evaluated in replicated trials conducted for each fungicide and assay type. The effective concentration to reduce mycelial growth or ascospore germination by 50% (EC50) was estimated for each isolate, fungicide, and assay type. The median EC50 values obtained from ascospore germination assays were 52.7, 10.0, and 2.7 times higher than those estimated from the mycelial growth for boscalid, fluazinam, and thiophanate-methyl, respectively. No significant correlation was found between EC50 values estimated by the two methods. These findings highlight differences that may be important in evaluating the sensitivity of S. sclerotiorum given the fungicide mode of action and how they will be used in the field.

Tracking Fungal Growth: Establishment of Arp1 as a Marker for Polarity Establishment and Active Hyphal Growth in Filamentous Ascomycetes.

Polar growth is a key characteristic of all filamentous fungi. It allows these eukaryotes to not only effectively explore organic matter but also interact within its own colony, mating partners, and hosts. Therefore, a detailed understanding of the dynamics in polar growth establishment and maintenance is crucial for several fields of fungal research. We developed a new marker protein, the actin-related protein 1 (Arp1) fused to red and green fluorescent proteins, which allows for the tracking of polar axis establishment and active hyphal growth in microscopy approaches. To exclude a probable redundancy with known polarity markers, we compared the localizations of the Spitzenkörper (SPK) and Arp1 using an FM4-64 staining approach. As we show in applications with the coprophilous fungus Sordaria macrospora and the hemibiotrophic plant pathogen Colletotrichum graminicola, the monitoring of Arp1 can be used for detailed studies of hyphal growth dynamics and ascospore germination, the interpretation of chemotropic growth processes, and the tracking of elongating penetration pegs into plant material. Since the Arp1 marker showed the same dynamics in both fungi tested, we believe this marker can be broadly applied in fungal research to study the manifold polar growth processes determining fungal life.

Abundant Small Protein ICARUS Inside the Cell Wall of Stress-Resistant Ascospores of Talaromyces macrosporus Suggests a Novel Mechanism of Constitutive Dormancy.

Ascospores of Talaromyces.macrosporus belong to the most stress resistant eukaryotic cells and show a constitutive dormancy, i.e., no germination occurs in the presence of rich growth medium. Only an extreme trigger as very high temperature or pressure is able to evoke synchronized germination. In this study, several changes within the thick cell wall of these cells are observed after a heat treatment: (i.) a change in its structure as shown with EPR and X-ray diffraction; (ii.) a release of an abundant protein into the supernatant, which is proportional to the extent of heat activation; (iii.) a change in the permeability of the cell wall as judged by fluorescence studies in which staining of the interior of the cell wall correlates with germination of individual ascospores. The gene encoding the protein, dubbed ICARUS, was studied in detail and was expressed under growth conditions that showed intense ascomata (fruit body) and ascospore formation. It encodes a small 7–14 kD protein. Blast search exhibits that different Talaromyces species show a similar sequence, indicating that the protein also occurs in other species of the genus. Deletion strains show delayed ascomata formation, release of pigments into the growth medium, higher permeability of the cell wall and a markedly shorter heat activation needed for activation. Further, wild type ascospores are more heat-resistant. All these observations suggest that the protein plays a role in dormancy and is related to the structure and permeability of the ascospore cell wall. However, more research on this topic is needed to study constitutive dormancy in other fungal species that form stress-resistant ascospores.

Rizobacterias con potencial antagonista in vitro a Mycosphaerella fijiensis Morelet

El empleo de bio-controladores en la agricultura beneficia los aspectos fisiológicos en plantas, a diferencia de la constante aplicación de pesticidas en el cultivo del banano ha ocasionado la pérdida de la sensibilidad en M. fijiensis, reduciendo la microbiota del suelo. El objetivo se enfocó en caracterizar el potencial antagónico de las PGPR en inhibición de germinación de ascósporas y desarrollo micelial de M. fijiensis. Se realizaron cultivos monospóricos de M. fijiensis e identificado por PCR. Se evaluaron los extractos celulares de Pseudomonas putida PB3-6, Klebsiella variicola BO3-4, Enterobacter asburiae BA4-19, Serratia marcescens PM3-8, Enterobacter asburiae PM3-14, Pseudomonas protegens CHA0, Pseudomonas fluorescens WCS417, Pseudomonas veronii R4 y Bacillus subtilis ATCC 5540 para sus evaluaciones antagonistas: a) Inhibición del tubo germinativo de las ascósporas al 2% y b) Desarrollo micelial al (2 y 10 %). La PCR empleado en la identificación de M. fijiensis se confirma el producto de amplificación de 1018 pb. El factor antagónico de los extractos celulares al 2 % de PM3-14 y CHA0 inhibe sobre el 80 % al desarrollo de los tubos germinativos. La inhibición al desarrollo micelial del extracto celular al 2 %, de CHA0 logró una efectividad del 54 % y las cepas (PM3-8, PM3-14 y BA4-19) con (32, 26 y 26 %). Al 10 % del extracto de la cepa PM3-8 inhibe el desarrollo micelial con niveles de turbidez de 0,47 (OD600nm). El empleo de estos bio-controladores en la agricultura ofrecerá una alternativa para beneficiar en la reducción del uso de agroquímicos

Global Sexual Fertility in the Opportunistic Pathogen Aspergillus fumigatus and Identification of New Supermater Strains.

A sexual cycle in Aspergillus fumigatus was first described in 2009 with isolates from Dublin, Ireland. However, the extent to which worldwide isolates can undergo sexual reproduction has remained unclear. In this study a global collection of 131 isolates was established with a near 1:1 ratio of mating types. All isolates were crossed to MAT1-1 or MAT1-2 Irish strains, and a subset of isolates from different continents were crossed together. Ninety seven percent of isolates were found to produce cleistothecia with at least one mating partner, showing that sexual fertility is not limited to the Irish population but is a characteristic of global A. fumigatus. However, large variation was seen in numbers of cleistothecia produced per cross, suggesting differences in the possibility for genetic exchange between strains in nature. The majority of crosses produced ascospores with >50% germination rates, but with wide variation evident. A high temperature heat shock was required to induce ascospore germination. Finally, a new set of highly fertile MAT1-1 and MAT1-2 supermater strains were identified and pyrimidine auxotrophs generated for community use. Results provide insights into the potential for the A. fumigatus sexual cycle to generate genetic variation and allow gene flow of medically important traits.

Antagonism of saprobe fungi from semiarid areas of the Northeast of Brazil against Sclerotinia sclerotiorum and biocontrol of soybean white mold

The antagonistic activity of 25 saprobe fungi from semiarid areas of Northeast Brazil was evaluated against Sclerotinia sclerotiorum (Lib.) de Bary (Helotiales: Sclerotiniaceae). Four fungi [Myrothecium sp. Tode (Hypocreales: Stachybotryaceae) isolate 2, Volutella minima Höhn. (Hypocreales: Nectriaceae), Phialomyces macrosporus P.C. Misra & P.H.B. Talbot (Pezizomycotina) and Dictyosporium tetraseriale Goh, Yanna & K.D. Hyde (Pleosporales: Dictyosporiaceae)] were selected and further tested their ability to inhibit mycelial growth, sclerotia formation and ascospore germination of S. sclerotiorum and to control white mold on soybean plants. V. minima and P. macrosporus filtrates at 50% effectively suppressed mycelial growth and Myrothecium sp. isolate 2 completely suppressed sclerotia formation and inhibited ascospore germination by over 95%, the same result as commercial fungicide fluazinam. Soybean plants pre-treated with Myrothecium sp. isolate 2, P. macrosporus, and V. minima and inoculated with S. sclerotiorum showed a reduction of 55.8%, 79.7%, and 83.2% of area under disease progress curve (AUDPC) of white mold, respectively, in relation to water. Collectively, these results underline the antagonistic activity of V. minima, P. macrosporus, and Myrothecium sp. isolate 2 against S. sclerotiorum and their potential as biocontrol agents of soybean white mold.

Crosstalk Between Pheromone Signaling and NADPH Oxidase Complexes Coordinates Fungal Developmental Processes.

Sexual and asexual development in filamentous ascomycetes is controlled by components of conserved signaling pathways. Here, we investigated the development of mutant strains lacking genes for kinases MAK2, MEK2, and MIK2, as well as the scaffold protein HAM5 of the pheromone response (PR) pathway. All had a defect in fruiting body development and hyphal fusion. Another phenotype was a defect in melanin-dependent ascospore germination. However, this deficiency was observed only in kinase deletion mutants, but not in strains lacking HAM5. Notably, the same developmental phenotypes were previously described for nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 (NOX1) mutants, but the germination defect was only seen in NOX2 mutants. These data suggest a molecular link between the pheromone signaling pathway and both NOX complexes. Using data from yeast two-hybrid (Y2H) analysis, we found that the scaffolding protein HAM5 interacts with NOR1, the regulator of NOX1 and NOX2 complexes. This interaction was further confirmed using differently fluorescent-labeled proteins to demonstrate that NOR1 and HAM5 co-localize at cytoplasmic spots and tips of mature hyphae. This observation was supported by phenotypic characterization of single and double mutants. The oxidative stress response and the initiation of fruiting bodies were similar in Δham5Δnor1 and Δham5, but distinctly reduced in Δnor1, indicating that the double deletion leads to a partial suppression of the Δnor1 phenotype. We conclude that the PR and NOX1 complexes are connected by direct interaction between HAM5 and NOR1. In contrast, PR kinases are linked to the NOX2 complex without participation of HAM5.

Effect of Wetness Duration and Incubation Temperature on Development of Ascosporic Infections by Sclerotinia sclerotiorum.

The impact of wetness duration and incubation temperatures on Sclerotinia sclerotiorum ascospore germination and ascosporic infection efficiency were evaluated. Ascospore germination was optimal when incubated in continuous moisture (free water) at 21°C. Significantly lower germination was observed at 10 or 30°C. Interrupting ascospore wet incubation was detrimental for germination. In infection efficiency studies, dry bean and canola flowers were inoculated with dry ascospores and placed on leaves of dry bean and canola plants, respectively. Dry bean plants were incubated for 196 h at 18 to 20°C in alternating 8 to 16 h wet/12 to 24 h dry periods. Canola plants were incubated for 240 h at 10, 15, 20, 25, or 30°C in alternating 6 to 18 h wet/18 to 6 h dry periods. Interrupting wet incubation delayed symptom appearance and hindered development of the epidemics on both plant types. Logistic regression models estimated at 50% the probability of disease development on dry bean and canola plants when 68 and 48 h of wet incubation at 20°C accumulated in a period of 6 days, respectively. The canola model was validated using data from field trials. Results of these studies will contribute to develop more accurate warning models for diseases caused by S. sclerotiorum.

Inoculum and Infection Dynamics of Polystigma amygdalinum in Almond Orchards in Spain.

Red leaf blotch (RLB) disease of almond, caused by Polystigma amygdalinum, is an important foliar disease in most production regions of the Mediterranean basin and the Middle East because severe infections may cause a premature defoliation of the tree. Some key aspects on the epidemiology of P. amygdalinum were studied in multiyear trials in two almond-growing regions in Spain, which included the seasonal development of perithecia and production and germination of ascospores along with the disease incubation and plant infectivity periods. Our results showed that primary inoculum was available in extended periods (January to August). Significant differences in ascospore amounts among regions, higher in the southern Andalusia and lower in the northern Catalonia, and years of study were detected. The factors geographical location, sampling period, and evaluation year were found significant on the development of P. amygdalinum perithecia. Variable ascospore germination rates were observed from April to July: >15% but rarely exceeding 30%. The RLB infectivity period in Catalonia extended from March to mid-June, whereas in Andalusia it was from March to May. The incubation period was mainly in a range of 5 to 10 weeks in Catalonia. The environmental conditions of October to January influence the available ascospore amounts in the next season. RLB infection occurs in spring to summer when mean temperatures are in the range 10 to 20°C. These results represent the first step in developing a prediction model of the disease that might serve as a tool for the control of RLB.

Phyllachora maydis Ascospore Release and Germination from Overwintered Corn Residue

Tar spot of corn, caused by Phyllachora maydis, has been reported in several upper Midwest states in the United States. This has led to expanded efforts to more thoroughly understand the biology of P. maydis and the epidemiology of tar spot. This study determined the potential for P. maydis ascospore release and germination from overwintered P. maydis-infected corn residues from various locations in the upper Midwest. Corn residue samples collected in the spring of 2019 from 12 fields in four states were examined. Ascospore release and germination were observed in all residue samples collected. The mean total number of ascospores released per milliliter of water ranged from 3.6 × 103 to 4.8 × 106 after 4-h incubation and 3.7 × 103 to 4.4 × 106 after 24-h incubation. The mean percent spore germination ranged from 0.7 to 24.1% after 4-h incubation and 2.4 to 24.9% after 24-h incubation. There was a significant inverse relationship between total numbers of ascospores released and the percent germination of the ascospores. Samples from Illinois consistently yielded the greatest total ascospore release but were also consistently among the samples with the lowest percent ascospore germination. Samples from Wisconsin and Indiana were among the lowest for total ascospore numbers but were among the highest for total ascospore germination. These findings provide evidence that P. maydis can overwinter in multiple areas of the upper Midwest. Future research should focus on reducing infested corn residue to reduce initial infection by P. maydis.

Use of Botanicals to Suppress Different Stages of the Life Cycle of Fusarium graminearum.

Fusarium head blight (FHB) is one of the most important cereal diseases worldwide, causing yield losses and contamination of harvested products with mycotoxins. Fusarium graminearum is one of the most common FHB-causing species in wheat and barley cropping systems. We assessed the ability of different botanical extracts to suppress essential stages of the fungal life cycle using three strains of F. graminearum (FG0410, FG2113, and FG1145). The botanicals included aqueous extracts from white mustard (Sinapis alba) seed flour (Pure Yellow Mustard [PYM] and Tillecur [Ti]) as well as milled Chinese galls (CG). At 2% concentration (wt/vol), PYM and Ti completely inhibited growth of mycelium of all F. graminearum strains whereas, at 1%, CG reduced the growth by 65 to 83%, depending on the strain. While PYM and Ti reduced the germination of both conidia and ascospores at 2% (wt/vol), CG was only effective in reducing conidia germination. Perithecia formation of FG0410 but not FG2113 was suppressed by all botanicals. Moreover, application of botanicals on mature perithecia led to a two- to fourfold reduction in discharge of ascospores. Using liquid chromatography (LC) with diode array detection, we quantified the principal glucosinolate component sinalbin of PYM and Ti. LC time-of-flight mass spectrometry was used to demonstrate that the bioactive matrix of CG contains different gallotannins as well as gallic and tannic acids. Possible antifungal mechanisms of the botanical matrices are discussed. The results of this study are promising and suggest that PYM, Ti, and CG should be explored further for efficacy at managing FHB.[Formula: see text] Copyright © 2019 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Protein compounds of Bacillus subtilis with in vitro antifungal activity against Pseudocercospora fijiensis (Morelet).

The metabolites of Bacillus subtilis CCIBP-M27 were evaluated as an antagonist of Pseudocercospora fijiensis. The culture filtrate did not inhibit ascospore germination but significantly reduced conidial germination and mycelial growth. Through microscopic analysis, deformations were observed as vacuolization and swelling in P. fijiensis mycelia when exposed to culture filtrate during 48h. A similar response was induced by peptide-type compounds found on Bacillus subtilis CCIBP-M27 culture filtrate. The results obtained suggest that the in vitro antifungal effect of the strain CCIBP-M27 against P. fijiensis is related to the action of diffused metabolites such as proteins or peptide substances.

A case study on the re-establishment of the cyanolichen symbiosis: where do the compatible photobionts come from?

In order to re-establish lichen symbiosis, fungal spores must first germinate and then associate with a compatible photobiont. To detect possible establishment limitations in a sexually reproducing cyanolichen species, we studied ascospore germination, photobiont growth and photobiont association patterns in Pectenia plumbea. Germination tests were made with ascospores from 500 apothecia under different treatments, and photobiont growth was analysed in 192 isolates obtained from 24 thalli. We determined the genotype identity [tRNALeu (UAA) intron] of the Nostoc cyanobionts from 30 P. plumbea thalli from one population. We also sequenced cyanobionts of 41 specimens of other cyanolichen species and 58 Nostoc free-living colonies cultured from the bark substrate. Not a single fungal ascospore germinated and none of the photobiont isolates produced motile hormogonia. Genetic analyses revealed that P. plumbea shares Nostoc genotypes with two other cyanolichen species of the same habitat, but these photobionts were hardly present in the bark substrate. Due to the inability of both symbionts to thrive independently, the establishment of P. plumbea seems to depend on Dendriscocaulon umhausense, the only cyanolichen species in the same habitat that reproduces asexually and acts as a source of appropriate cyanobionts. This provides support to the hypothesis about facilitation among lichens.

Phylogenetic and Morphological Reassessment of Mycosphaerella nawae, the Causal Agent of Circular Leaf Spot in Persimmon.

Persimmon (Diospyros kaki) fruit production is severely affected by circular leaf spot worldwide. Mycosphaerella nawae causes circular leaf spot of persimmon (CLSP) and can result in leaf spot, defoliation, early fruit maturation, and subsequent softening and abscission. The morphology and phylogenetic position of M. nawae within the family Mycosphaerellaceae is, therefore, of utmost importance given its impact on persimmon production. Based on previous morphological and molecular studies, the phylogenetic position of the anamorphic genera associated with M. nawae remain in confusion. In the present study, 15 isolates of M. nawae were collected from the tissue of living leaves exhibiting leaf spot symptoms. A subsample of three isolates was characterized phylogenetically and morphologically. Isolates were compared based on DNA sequence data for the internal transcribed spacer region (ITS1-5.8S ITS2), part of the 28S nrDNA including domains D1-D3 (LSU), actin (Act), translation elongation factor 1-alpha (EF-1α), and RNA polymerase II second largest subunit (rpb2). The anamorph and teleomorph structures, ascospore germination patterns, as well as host specificity were used to describe the isolates. The phylogenetic and morphological analyses revealed that M. nawae requires a new holomorphic genus within Mycosphaerellaceae, described herein as Plurivorosphaerella gen. nov. A host specificity test revealed that Plurivorosphaerella nawae comb. nov. (M. nawae) can superficially colonize, but not infect, apple, peach, cherry, and plum.