Airborne ascospore discharge with co-dispersal of attached epihymenial algae in some foliicolous lichens.

PREMISE: Lichen-forming fungi that colonize leaf surfaces must find a compatible algal symbiont, establish lichen symbiosis, and reproduce within the limited life span of their substratum. Many produce specialized asexual propagules that appear to be dispersed by rain and runoff currents, but less is known about dispersal of their meiotic ascospores. In some taxa, a layer of algal symbionts covers the hymenial surface of the apothecia, where asci discharge their ascospores. We examined the untested hypothesis that their ascospores are ejected into air currents and carry with them algal symbionts from the epihymenial layer for subsequent lichenization. METHODS: Leaves bearing the lichens Calopadia puiggarii, Sporopodium marginatum (Pilocarpaceae), and Gyalectidium viride (Gomphillaceae) were collected in southern Florida. The latter two species have epihymenial algal layers. Leaf fragments with apotheciate thalli were affixed in petri dishes, with glass cover slips attached inside the lid over the thalli. Subsequent discharge of ascospores and any co-dispersed algae was evaluated with light microscopy. RESULTS: All three species discharged ascospores aerially. Discharged ascospores were frequently surrounded by a halo-like sheath of transparent material. In the two species with an epihymenial algal layer, most dispersing ascospores (>90%) co-transported algal cells attached to the spore sheath or wall. CONCLUSIONS: While water may be the usual vector for their asexual propagules, foliicolous lichen-forming fungi make use of air currents to disperse their ascospores. The epihymenial algal layer represents an adaptation for efficient co-dispersal of the algal symbiont with the next genetic generation of the fungus.

The C2H2 Transcription Factor SsZFH1 Regulates the Size, Number, and Development of Apothecia in Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a notorious phytopathogenic Ascomycota fungus with a host range of >600 plant species worldwide. This homothallic Leotiomycetes species reproduces sexually through a multicellular apothecium that produces and releases ascospores. These ascospores serve as the primary inoculum source for disease initiation in the majority of S. sclerotiorum disease cycles. The regulation of apothecium development for this pathogen and other apothecium-producing fungi remains largely unknown. Here, we report that a C2H2 transcription factor, SsZFH1 (zinc finger homologous protein), is necessary for the proper development and maturation of sclerotia and apothecia in S. sclerotiorum and is required for the normal growth rate of hyphae. Furthermore, ΔSszfh1 strains exhibit decreased H2O2 accumulation in hyphae, increased melanin deposition, and enhanced tolerance to H2O2 in the process of vegetative growth and sclerotia formation. Infection assays on common bean leaves, with thin cuticles, and soybean and tomato leaves, with thick cuticles, suggest that the deletion of Sszfh1 slows the mycelial growth rate, which in turn affects the expansion of leaf lesions. Collectively, our results provide novel insights into a major fungal factor mediating maturation of apothecia with additional effects on hyphae and sclerotia development.

Population genomics of a reindeer lichen species from North American lichen woodlands.

PREMISE: Lichens are one of the main structural components of plant communities in the North American boreal biome. They play a pivotal role in lichen woodlands, a large ecosystem situated north of the closed-crown forest zone, and south of the forest-tundra zone. In Eastern Canada (Quebec), there is a remnant LW found 500 km south of its usual distribution range, in the Parc National des Grands-Jardins, originated mainly because of wildfires. We inferred the origin of the lichen Cladonia stellaris from this LW and assessed its genetic diversity in a postfire succession. METHODS: We genotyped 122 individuals collected across a latitudinal gradient in Quebec. Using the software Stacks, we compared four different approaches of locus selection and single-nucleotide polymorphism calling. We identified the best fitting approach to investigate population structure and estimate genetic diversity of C. stellaris. RESULTS: Populations in southern Quebec are not genetically different from those of northern LWs. The species consists of at least four phylogenetic lineages with elevated levels of genetic diversity and low co-ancestry. In Parc National des Grands-Jardins, we reported high values of genetic diversity not related with time since fire disturbance and low genetic differentiation among populations with different fire histories. CONCLUSIONS: This first population genomic study of C. stellaris is an important step forward to understand the origin and biogeographic patterns of lichen woodlands in North America. Our findings also contribute to the understanding of the effect of postfire succession on the genetic structure of the species.

Carpogenic Germinability of Diverse Sclerotinia sclerotiorum Populations Within the Southwestern Australian Grain Belt.

Sclerotinia stem rot, caused by the necrotrophic plant pathogen Sclerotinia sclerotiorum (Lib.) de Bary, is a major disease of canola and pulses in Australia. Current disease management relies greatly on cultural and chemical means of control. Timing of fungicide applications remains a challenge, because efficacy is dependent on accurate prediction of ascospore release and presence on the plant. The aims of this study were to determine the optimal temperature for carpogenic germination of S. sclerotiorum populations sampled from canola and lupin fields in southwestern Australia and characterize diversity using mycelial compatibility groupings (MCGs). Sclerotia were collected from four diseased canola and one diseased lupin field from across southwestern Australia. Forty sclerotia from each population were incubated at four alternating temperatures of 30/15, 20/15, 20/4, and 15/4°C (12-h/12-h light/dark cycle) and assessed every 2 to 3 days for a 180-day period. MCG groupings for populations were characterized using 12 reference isolates. Results indicated the time to initial carpogenic germination decreased as diurnal temperature fluctuations decreased, with a fluctuation of 5°C (20/15°C) having the most rapid initial germination followed by 11°C (15/4°C) followed by 16°C (20/4°C). Optimal germination temperature for all five populations was 20/15°C; however, population responses to other diurnal temperature regimes varied considerably. No germination was observed at 30/15°C. MCG results indicate extensive diversity within and between populations, with at least 40% of sclerotia within each population unable to be characterized. We suggest that this diversity has enabled S. sclerotiorum populations to adapt to varying environmental conditions within southwestern Australia.

Escape from the cryptic species trap: lichen evolution on both sides of a cyanobacterial acquisition event.

Large, architecturally complex lichen symbioses arose only a few times in evolution, increasing thallus size by orders of magnitude over those from which they evolved. The innovations that enabled symbiotic assemblages to acquire and maintain large sizes are unknown. We mapped morphometric data against an eight-locus fungal phylogeny across one of the best-sampled thallus size transition events, the origins of the Placopsis lichen symbiosis, and used a phylogenetic comparative framework to explore the role of nitrogen-fixing cyanobacteria in size differences. Thallus thickness increased by >150% and fruiting body core volume increased ninefold on average after acquisition of cyanobacteria. Volume of cyanobacteria-containing structures (cephalodia), once acquired, correlates with thallus thickness in both phylogenetic generalized least squares and phylogenetic generalized linear mixed-effects analyses. Our results suggest that the availability of nitrogen is an important factor in the formation of large thalli. Cyanobacterial symbiosis appears to have enabled lichens to overcome size constraints in oligotrophic environments such as acidic, rain-washed rock surfaces. In the case of the Placopsis fungal symbiont, this has led to an adaptive radiation of more than 60 recognized species from related crustose members of the genus Trapelia. Our data suggest that precyanobacterial symbiotic lineages were constrained to forming a narrow range of phenotypes, so-called cryptic species, leading systematists until now to recognize only six of the 13 species clusters we identified in Trapelia.

The Parmotrema acid test: a look at species delineation in the P. perforatum group 40 y later.

Parmotrema perforatum and its relatives form a morphologically distinctive group of species, most of which are common and endemic to eastern North America. Species delimitation in this ecologically important group was the subject of extensive inquiry before the advent of molecular systematics and computationally intensive niche modeling. As part of a large-scale lichen biodiversity inventory of the Mid-Atlantic Coastal Plain, we used ITS sequence data to examine the utility of characters (morphological, chemical, reproductive, ecological) in circumscribing four species in this group (P. hypoleucinum, P. hypotropum, P. perforatum, P. subrigidum). We found that P. hypoleucinum and P. subrigidum as currently circumscribed are monophyletic and the latter comprises two chemotypes differing in the presence or absence of norstictic acid in addition to alectoronic acid. The sequences of P. hypotropum and P. perforatum, which are chemically identical species and differ only in reproductive mode, were intermixed in a single, well-supported clade. The two chemotypes of P. subrigidum are partially allopatric and their sequences are >99% identical. Nonetheless, niche modeling suggests they occupy significantly different ecological niches. These results provide a new perspective on much-debated questions on species circumscription in lichens and suggest new avenues for genetic, ecological and systematic research.

Mummy berry pseudosclerotia survive for several years.

Organic blueberry production in the PNW has many challenges, including diseases like mummy berry caused by the fungus Monilinia vaccinii-corymbosi (Reade) Honey. Management recommendations focus on reducing overwintering pseudosclerotia, however, it is unknown how long they survive. Based on qualitative observations pseudosclerotia are hypothesized to survive multiple years after contact with the soil surface. The development of apothecia from M. vaccinii-corymbosi pseudosclerotia was evaluated over multiple years at a location without a history of blueberry production. A total of 1,000 pseudosclerotia were placed on field soil plots in 2018 and replicated eight times. Another 100 pseudosclerotia were placed in wire corrals on field soil and replicated fifteen times. Plots and corrals were regularly examined each spring for the emergence of apothecia. The pseudosclerotia were able to survive, germinate, and produce apothecia for up to five years after their placement. Very few pseudosclerotia produced apothecia in any year, varying from 0 to 18 at any observed time. Pieces of partial or whole pseudosclerotia were observed for up to three years after placement. Our study shows that a pseudosclerotial "seed bank" exists under blueberry bushes, necessitating a long-term implementation of mummy berry cultural management tactics.

The phylogeny and taxonomy of Upretia (Caloplacoideae, Teloschistaceae), reveal three new species from Southwestern China.

Several specimens of Upretia from Southwest China are morphologically and phylogenetically distinct from currently recognized species in the genus. These specimens are here accommodated within a new species, Upretiazeorina Li J. Li & Printzen. It is characterized by an areolate to squamulose thallus with brown to blackish brown upper surface, pruinose, zeorine type apothecia, black discs, narrowly bacilliform conidia, and the production of gyrophoric acid. Two other specimens of Upretia from China are distinct from currently accepted species and tentatively referred to as Upretia sp. 1 and Upretia sp. 2. A key to all known species of Upretia is also provided.

Apothecial Ancestry, Evolution, and Re-Evolution in Thelebolales (Leotiomycetes, Fungi).

Closed cleistothecia-like ascomata have repeatedly evolved in non-related perithecioid and apothecioid lineages of lichenized and non-lichenized Ascomycota. The evolution of a closed, darkly pigmented ascoma that protects asci and ascospores is conceived as either an adaptation to harsh environmental conditions or a specialized dispersal strategy. Species with closed ascomata have mostly lost sterile hymenial elements (paraphyses) and the capacity to actively discharge ascospores. The class Leotiomycetes, one of the most speciose classes of Ascomycota, is mainly apothecioid, paraphysate, and possesses active ascospore discharge. Lineages with closed ascomata, and their morphological variants, have evolved independently in several families, such as Erysiphaceae, Myxotrichaceae, Rutstroemiaceae, etc. Thelebolales is a distinctive order in the Leotiomycetes class. It has two widespread families (Thelebolaceae, Pseudeurotiaceae) with mostly closed ascomata, evanescent asci, and thus passively dispersed ascospores. Within the order, closed ascomata dominate and a great diversity of peridia have evolved as adaptations to different dispersal strategies. The type genus, Thelebolus, is an exceptional case of ascomatal evolution within the order. Its species are the most diverse in functional traits, encompassing species with closed ascomata and evanescent asci, and species with open ascomata, active ascospore discharge, and paraphyses. Open ascomata were previously suggested as the ancestral state in the genus, these ascomata depend on mammals and birds as dispersal agents. In this scheme, species with closed ascomata, a lack of paraphyses, and passive ascospore discharge exhibit derived traits that evolved in adaptation to cold ecosystems. Here, we used morphological and phylogenetic methods, as well as the reconstruction of ancestral traits for ascomatal type, asci dehiscence, the presence or absence of paraphyses, and ascospore features to explore evolution within Thelebolales. We demonstrate the apothecial ancestry in Thelebolales and propose a new hypothesis about the evolution of the open ascomata in Thelebolus, involving a process of re-evolution where the active dispersal of ascospores appears independently twice within the order. We propose a new family, Holwayaceae, within Thelebolales, that retains the phenotypic features exhibited by species of Thelebolus, i.e., pigmented capitate paraphyses and active asci discharge with an opening limitation ring.

Revision of Immersaria and a new lecanorine genus in Lecideaceae (lichenised Ascomycota, Lecanoromycetes).

The species Immersariacupreoatra has been included in Bellemerea. This caused us to reconsider the relationships between Bellemerea and the lecanorine species of Immersaria and to question the monophyly of Immersaria. Amongst 25 genera of the family Lecideaceae, most have lecideine apothecia, the exceptions being Bellemerea and Koerberiella, which have lecanorine apothecia. According to previous classifications, Immersaria included species with both lecanorine and lecideine apothecia. A five-loci phylogenetic tree (nrITS, nrLSU, RPB1, RPB2, and mtSSU) for Lecideaceae showed that Immersaria was split into two clades: firstly, all the lecideine apotheciate species and secondly, all the lecanorine apotheciate species. The latter clade was closely related to the remaining lecanorine apotheciate genera: Bellemerea and Koerberiella. Therefore, the genus concept of Immersaria is revised accordingly and a new lecanorine genus Lecaimmeria is proposed. Furthermore, four new species for Immersaria and seven new species and three new combinations for the new genus Lecaimmeria are proposed. Keys to Immersaria and the new genus Lecaimmeria are provided.

Stem and Crown Rot of Chickpea in California Caused by Sclerotinia trifoliorum.

The identities of Sclerotinia isolates obtained from chickpea plants showing stem and crown rot were determined using morphological characteristics, variations in group I introns, and internal transcribed spacer (ITS) sequences. Isolates could be separated into two groups based on growth rates at 22°C, fast growing (about 40 mm per day) versus slow growing (about 20 mm per day). All fast-growing isolates induced stronger color change of a pH-indicating medium than did slow-growing isolates at 22°C. The slow-growing isolates contained at least one group I intron in the nuclear small subunit rDNA, whereas all fast-growing isolates lacked group I introns in the same DNA region. ITS sequences of the slow-growing isolates were identical to sequences of Sclerotinia trifoliorum. Those of the fast-growing isolates were identical to sequences of S. sclerotiorum. Finally, the slow-growing isolates showed ascospore dimorphism, a definitive character of S. trifoliorum, whereas the fast-growing isolates showed no ascospore dimorphism. Isolates of both species were pathogenic on chickpea and caused symptoms similar to those observed in the field. This study not only associated the differences between S. sclerotiorum and S. trifoliorum in growth rates, group I introns, ITS sequences, and ascospore morphology, but also represented the first report that S. trifoliorum causes stem and crown rot of chickpea in North America.

Tissue-specific localization of polyketide synthase and other associated genes in the lichen, Cladonia rangiferina, using laser microdissection.

The biosynthesis of two polyketides, atranorin and fumarprotocetraric acid, produced from a lichen-forming fungus, Cladonia rangiferina (L.) F. H. Wigg. was correlated with the expression of eight fungal genes (CrPKS1, CrPKS3, CrPKS16, Catalase (CAT), Sugar Transporter (MFsug), Dioxygenase (YQE1), C2H2 Transcription factor (C2H2), Transcription Factor PacC (PacC), which are thought to be involved in polyketide biosynthesis, and one algal gene, NAD-dependent deacetylase sirtuin 2 (AsNAD)), using laser microdissection (LMD). The differential gene expression levels within the thallus tissue layers demonstrate that the most active region for potential polyketide biosynthesis within the lichen is the outer apical region proximal to the photobiont but some expression also occurs in reproductive tissue. This is the first study using laser microdissection to explore gene expression of these nine genes and their location of expression; it provides a proof-of-concept for future experiments exploring tissue-specific gene expression within lichens; and it highlights the utility of LMD for use in lichen systems.

Vegetative compatibility groups and sexual reproduction among Spanish Monilinia fructicola isolates obtained from peach and nectarine orchards, but not Monilinia laxa.

The frequency of occurrence of Monilinia fructicola in the Ebro Valley, Spain has increased since its first appearance in 2006, and M. fructicola has displaced Monilinia laxa, the native species which is the main cause of brown rot in peaches in this valley. In order to determine the characteristics that may be related to the displacement, we studied the capacity to generate new genotypic combinations of M. fructicola under laboratory conditions. The morphology and parasitic ability from ten field isolates of M. fructicola and M. laxa collected from three different orchards in the valley, and sampling from five different lesions were studied. Nitrate-non-utilising (nit) mutants were generated in order to test the isolates for vegetative compatibility which was done by assessing their colony growth when cultured singly or in pairs on media that contained different nitrogen sources. For the M. fructicola isolates, five vegetative compatibility groups (VCGs) were identified using the nit mutants and six VCGs were identified when they were grown on potato dextrose agar dishes. In all instances, the vegetatively compatible M. fructicola isolates came mainly from the same orchard. Only one VCG displays the same morphological and competition characteristics. No VCGs were identified among the M. laxa isolates. We did not find any apothecia of M. laxa and M. fructicola isolates in the soil of the three orchards, but we were able to produce apothecia of M. fructicola in the laboratory. Our finding of sexual reproduction and VCGs in the M. fructicola isolates suggests that the genetic variability of M. fructicola could be maintained by sexual and/or parasexual recombination.

Influência de herbicidas e fungicidas na germinação carpogênica de escleródios de Sclerotinia sclerotiorum (Lib. ) de Bary / Influence of herbicides and fungicides in the carpogenic germination of sclerotia of Sclerotinia sclerotiorum (Lib. ) de Bary

Sclerotinia sclerotiorum é um fungo polífago apresentando mais de 400 espécies hospedeiras. No Brasil relata-se que sua ocorrência tenha aumentado a partir da safra 2003/2004 em função do uso de sementes infectadas, falta de rotação de culturas, entre outras causas. Nos EUA, há relatos que o aumento da doença também tenha ocorrido devido à redução no uso de herbicidas do grupo das triazinas na cultura do milho, em rotação. Alguns trabalhos relatam que esse herbicida atua na formação de apotécios anormais e na inibição da produção de ascósporos. Frente a isso o presente trabalho objetivou o estudo do efeito de 12 herbicidas e 5 fungicidas sobre a germinação carpogênica de S. sclerotiorum. O trabalho foi conduzido em laboratório na Universidade Estadual de Ponta Grossa. Em caixas tipo gerbox foram colocados 80 gramas de substrato e 20 escleródios. A aplicação foi realizada utilizando um volume de calda de 400 L ha-1. Após a aplicação as caixas foram acondicionadas em ambiente controlado (20°C e fotoperíodo de 12 horas). Os tratamentos utilizados foram: glifosato, lactofen, atrazina, setoxidim, diuron, diuron + paraquat, imazaquim, trifluralina, diclofop metílico, clorimuron etílico, metribuzim e imazetapir (herbicidas) e fluazinam, procimidone, carbendazim, dimoxystrobin + boscalid e tiofanato metílico (fungicidas). Aos 36 dias após a aplicação foi realizada uma contagem do número de escleródios germinados, número de estipes e número de apotécios por escleródio. Para os dados de germinação de escleródios, foi constatado que somente dimoxystrobin + boscalid apresentou germinação inferior a 70%. Os demais tratamentos apresentaram germinação superior a 83%. Para número de estipes formadas por escleródio foi constatado que todos os tratamentos apresentaram de duas a quatro estipes por escleródio. Foi observado que os tratamentos com dimoxystrobin + boscalid, tiofanato metílico e carbendazim apresentaram as menores taxas de diferenciação de estipes em apotécios, ou seja, constatou-se a formação de estipes inviáveis. No tratamento com dimoxystrobin + boscalid foi constatado uma viabilidade de apenas 34,56% de suas estipes. Os resultados aqui apresentados mostram que a aplicação antecipada desses tratamentos pode ser realizada visando atingir a fonte de inóculo (escleródios) no solo, reduzindo o potencial de infecção dos mesmos e consequentemente sua incidência sobre culturas.

Sclerotinia sclerotiorum is a pathogenic fungus with over 400 host species. In Brazil, it is reported that its occurrence has increased since the 2003-2004 growing season due to use of infected seed, lack of crop rotation, and several other factors. In the United States, reports suggest that the disease increase has occurred due to reduced use of herbicides from the triazine group on corn in rotation with soybean. Reports show that these herbicides affect S. sclerotiorum, causing the formation of abnormal apothecia and inhibiting ascospore production. Because of the possible linkage with herbicide use, a study was designed to examine the effect of 12 herbicides and 5 fungicides on the carpogenic germination of S. sclerotiorum. This work was performed at Universidade Estadual de Ponta Grossa. Eighty grams of sterilized field soil and 20 sclerotia, placed on the soil surface, were added to "gerbox" boxes. Chemicals were applied once using a rate of 400 L ha-1. After application, boxes were incubated in a controlled environment (20°C with a photoperiod of 12 hours). The treatments used were: glyphosate, lactofen, atrazine, sethoxydim, diuron, diuron + paraquat, imazaquin, trifluralin, diclofop methyl, chrlorimuron ethyl, metribuzin and imazethapyr (herbicides) and fluazinam, procymidone, carbendazim, dimoxystrobin + boscalid and thiophanate methyl (fungicides). The number of germinated sclerotia, stipes and apothecia was recorded 36 days after chemical application. For sclerotia germination, only dimoxystrobin + boscalid reduced germination below 70%. All other treatments had greater than 83% sclerotia germination. The number of stipes produced per sclerotium was constant for all treatments (two to four stipes per sclerotium). Dimoxystrobin + boscalid, thiophanate methyl and carbendazim had the lowest level of apothecia formation, which means inviable stipes were formed. For the treatment of dimoxystrobin + boscalid, only 34.56% of stipes developed into mature apothecia. The results of this study demonstrate that application of herbicides did not affect the sclerotia of S. sclerotiorum. Three of the five fungicide tested in this trial, dimoxystrobin + boscalid, thiophanate methyl and carbendazim, can reduce inoculum on the soil, which, in turn, would likely reduce disease incidence in the following crop.