Inhibitory effects of essential oils from Asteraceae plant against pathogenic fungi of Panax notoginseng.

AIMS: Diseases caused by pathogenic fungi was a major constrain in increasing productivity and improving quality of Panax notoginseng. The aim of this research was to evaluate the inhibitory activity of essential oils (EOs) from Asteraceae family, Chrysanthemum indicum and Laggera pterodonta, against pathogenic fungi of P. notoginseng. METHODS AND RESULTS: The antifungal activity was investigated using multiple methods, disclosing that the EOs from C. indicum and L. pterodonta are active against hypha growth of different fungi but with different degrees of potency. Checkerboard testing indicated that the combination of EOs with hymexazol had synergistic effect against Pythium aphanidermatum, and exhibited additive effects against bulk of targeted pathogenic fungi. Besides, we found that the baseline sensitivity of Fusarium oxysporum to L. pterodonta EOs was higher than those of C. indicum by means of mycelium growth rate method. Finally, the practicability of those EOs as plant pesticide was confirmed by in vivo model showing that EOs can significantly inhibit the occurrence of root rot of P. notoginseng caused by F. oxysporum. CONCLUSION: Those studies suggest that the EOs from C. indicum and L. pterodonta had the potential to develop into new pollution-free pesticides for the protection of precious Chinese herbal medicines. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provided a new way of biological control for overcoming the frequent diseases occurrence of P. notoginseng.

Molecular evidence supports simultaneous association of the achlorophyllous orchid Chamaegastrodia inverta with ectomycorrhizal Ceratobasidiaceae and Russulaceae.

BACKGROUND: Achlorophyllous orchids are mycoheterotrophic plants, which lack photosynthetic ability and associate with fungi to acquire carbon from different environmental sources. In tropical latitudes, achlorophyllous forest orchids show a preference to establish mycorrhizal relationships with saprotrophic fungi. However, a few of them have been recently found to associate with ectomycorrhizal fungi and there is still much to be learned about the identity of fungi associated with tropical orchids. The present study focused on mycorrhizal diversity in the achlorophyllous orchid C. inverta, an endangered species, which is endemic to southern China. The aim of this work was to identify the main mycorrhizal partners of C. inverta in different plant life stages, by means of morphological and molecular methods. RESULTS: Microscopy showed that the roots of analysed C. inverta samples were extensively colonized by fungal hyphae forming pelotons in root cortical cells. Fungal ITS regions were amplified by polymerase chain reaction, from DNA extracted from fungal mycelia isolated from orchid root samples, as well as from total root DNA. Molecular sequencing and phylogenetic analyses showed that the investigated orchid primarily associated with ectomycorrhizal fungi belonging to a narrow clade within the family Ceratobasidiaceae, which was previously detected in a few fully mycoheterotrophic orchids and was also found to show ectomycorrhizal capability on trees and shrubs. Russulaceae fungal symbionts, showing high similarity with members of the ectomycorrhizal genus Russula, were also identified from the roots of C. inverta, at young seedling stage. Ascomycetous fungi including Chaetomium, Diaporthe, Leptodontidium, and Phomopsis genera, and zygomycetes in the genus Mortierella were obtained from orchid root isolated strains with unclear functional role. CONCLUSIONS: This study represents the first assessment of root fungal diversity in the rare, cryptic and narrowly distributed Chinese orchid C. inverta. Our results provide new insights on the spectrum of orchid-fungus symbiosis suggesting an unprecedented mixed association between the studied achlorophyllous forest orchid and ectomycorrhizal fungi belonging to Ceratobasidiaceae and Russulaceae. Ceratobasidioid fungi as dominant associates in the roots of C. inverta represent a new record of the rare association between the identified fungal group and fully mycoheterotrophic orchids in nature.

Three new Leptographium spp. (Ophiostomatales) infecting hardwood trees in Norway and Poland.

Species of Leptographium are characterized by mononematous or synnematous conidiophores and are commonly associated with different arthropods. Some of them also produce a sexual state characterised by globose ascomata with elongated necks. Compared to investigations on coniferous trees, the occurrence of Leptographium species on hardwood trees has been poorly studied in Europe. During a survey of ophiostomatoid fungi on various hardwood tree species in Norway and Poland, three unusual species, which fit in the broader morphological description of Leptographium spp., were found in association with Trypodendron domesticum, Trypodendron signatum and Dryocoetes alni, and from wounds on a variety of hardwoods. Phylogenetic analyses of sequence data for six different loci (ITS1-5.8 S-ITS2, ITS2-LSU, ACT, ß-tubulin, CAL, and TEF-1α) showed that these Leptographium species are phylogenetically closely related to the species of the Grosmannia olivacea complex. The first species forms a well-supported lineage that includes Ophiostoma brevicolle, while the two other new taxa resided in a separate lineage; possibly affiliated with Grosmannia francke-grosmanniae. All the new species produce perithecia with necks terminating in ostiolar hyphae and orange-section shaped ascospores with cucullate, gelatinous sheaths. These species also produce dark olivaceous mononematous asexual states in culture. In addition, two of the newly described species have a second type of conidiophore with a short and non-pigmented stipe. The new Leptographium species can be easily distinguished from each other by their appearance and growth in culture. Based on novel morphological characters and distinct DNA sequences, these fungi were recognised as new taxa for which the names Leptographium tardum sp. nov., Leptographium vulnerum sp. nov., and Leptographium flavum sp. nov. are provided.

[Biological and epidemiological characteristics of the pathogen of hypertrophy sorosis scleroteniosis, Ciboria shiraiana].

Objective: We studied the biological and the epidemiological characteristics of the pathogen of hypertrophy sorosis scleroteniosis, which is a devastating fungal disease of mulberry. Methods: We studied the asexual and sexual reproductive phase stages of C. shiraiana, including the infection ability of hyphal, dormancy of sclerotia, the structures, release, number and germination of ascospores from apothecia, as well as the phenology of sclerotial germination. Results: In C. shiraiana, hyphae had no infection ability toward the female flowers of mulberry. Sclerotia of C. shiraiana must undergo cold treatment above 6 weeks, then the dormancy-breaking sclerotia could germinate to apothecia. One to fifteen apothecia were germinated from one sclerotium, and the number of ascospores in a 1.5 cm diameter apothecia could contain up to (5.6-6.3)×107. Ascospore C. shiraiana had significantly higher germination rates in acid than in neutral and alkaline environments. From late January to middle April, sclerotia germinated to apothecia, and got the highest value in the middle of March. Conclusion: C. shiraiana is a formidable pathogen to cause epidemic disease and damage in mulberry.

[Micro-morphological variation pattern of Isaria cicadae].

Objective: To reveal the morphological pattern of Isaria cicadae. Methods: We observed 17 morphological characters and measured 75 strains of 15 populations in I. cicadae. Statistical analysis system (SAS) 8.1 was used to analyze the morphological data, the morphological pattern was analyzed in 15 populations of I. cicadae, using the descriptive statistical analysis, nested analysis and Q cluster analysis. Results: Two types of asexual conidium (large and small conidium) were observed in I. cicadae. The gourd-shaped and bottle-shaped conidiogenous cells were observed in I. cicadae. Many chlamydospores of I. cicadae were easy to form in PDA medium. Many fusion hyphae were generated between hyphae, and some fusion hyphae between hypha and chlamydospore, the fusion hyphae between conidiogenous cells were also observed. The CV of 17 morphological characters was from 13.07 to 104.09% in I. cicadae, indicating an ample morphological diversity at the species level. The nested variation analysis of the 17 morphological characters indicated that about 11.29% of the variability was attributable to the differentiation among populations, the rest 15.27% of the variability was derived from individual strains, and the remaining 73.44% was resided in the observations in the same strain. Conclusion: The phenotypic variation within strain was the main morphological variation of I. cicadae. The morphological characters had no significant relationship with geographical origin in I. cicadae.

Different levels of hyphal self-incompatibility modulate interconnectedness of mycorrhizal networks in three arbuscular mycorrhizal fungi within the Glomeraceae.

Arbuscular mycorrhizal fungi (AMF) live in symbiosis with most plant species and produce underground extraradical hyphal networks functional in the uptake and translocation of mineral nutrients from the soil to host plants. This work investigated whether fungal genotype can affect patterns of interconnections and structural traits of extraradical mycelium (ERM), by comparing three Glomeraceae species growing in symbiosis with five plant hosts. An isolate of Funneliformis coronatus consistently showed low ability to form interconnected ERM and self-incompatibility that represented up to 21% of hyphal contacts. The frequency of post-fusion self-incompatible interactions, never detected before in AMF extraradical networks, was 8.9%. In F. coronatus ERM, the percentage of hyphal contacts leading to perfect hyphal fusions was 1.2-7.7, while it ranged from 25.8-48 to 35.6-53.6 in Rhizophagus intraradices and Funneliformis mosseae, respectively. Low interconnectedness of F. coronatus ERM resulted also from a very high number of non-interacting contacts (83.2%). Such findings show that AMF genotypes in Glomeraceae can differ significantly in anastomosis behaviour and that ERM interconnectedness is modulated by the fungal symbiont, as F. coronatus consistently formed poorly interconnected networks when growing in symbiosis with five different host plants and in the asymbiotic stage. Structural traits, such as extent, density and hyphal self-compatibility/incompatibility, may represent key factors for the differential performance of AMF, by affecting fungal absorbing surface and foraging ability and thus nutrient flow from soil to host roots.

Contribution of arbuscular mycorrhizal fungi of sedges to soil aggregation along an altitudinal alpine grassland gradient on the Tibetan Plateau.

The diversity of arbuscular mycorrhizal fungi (AMF) in sedges on the Tibetan Plateau remains largely unexplored, and their contribution to soil aggregation can be important in understanding the ecological function of AMF in alpine ecosystems. Roots of Kobresia pygmaea C.B. Clarke and Carex pseudofoetida Kük. in alpine Kobresia pastures along an elevational transect (4149-5033 m) on Mount Mila were analysed for AMF diversity. A structural equation model was built to explore the contribution of biotic factors to soil aggregation. Sedges harboured abundant AMF communities covering seven families and some operational taxonomic units are habitat specific. The two plant species hosted similar AMF communities at most altitudes. The relative abundance of the two sedges contributed largely to soil macroaggregates, followed by extraradical mycorrhizal hyphae (EMH) and total glomalin-related soil protein (T-GRSP). The influence of plant richness was mainly due to its indirect influence on T-GRSP and EMH. There was a strong positive correlation between GRSP and soil total carbon and nitrogen. Our results indicate that mycorrhization might not be a major trait leading to niche differentiation of the two co-occurring sedge species. However, AMF contribute to soil aggregation and thus may have the potential to greatly influence C and N cycling in alpine grasslands.

A new species of Meliolinites associated with Buxus leaves from the Oligocene of Guangxi, southern China.

A new species of Meliolinites (fossil Meliolaceae), M. buxi sp. nov., is reported from the Oligocene Ningming Formation of Guangxi, South China. The fungus has hyphopodia characteristics of extant Meliolaceae, such as thick-walled, branching hyphae with appressoria and phialides. However, these fossils entirely lack mycelial or perithecial setae and have only a few phialides, thereby distinguishing the new species from most known species. The fungus was discovered on the adaxial and abaxial cuticles of several fossilized Buxus leaves. Thickening and twisting of cell walls in the Buxus leaf cuticle, along with the parasitic feeding strategy of the extant Meliolaceae, suggest that a parasitic interaction between Buxus and M. buxi seems feasible. The distribution of modern Meliolaceae suggests that they live in warm, humid subtropical-tropical climates. It is possible that the presence of M. buxi indicates a similar climatic condition. The co-occurrence of large-leaf Buxus and floristic comparisons of the Ningming assemblage also corroborate this conclusion.

Thelephora versatilis and Thelephora pseudoversatilis: two new cryptic species with polymorphic basidiomes inhabiting tropical deciduous and sub-perennial forests of the Mexican Pacific coast.

Thelephora is a genus of ectomycorrhizal basidiomycetes with basidiomes of varied shape which has been poorly studied in tropical ecosystems. In this paper, we present Thelephora versatilis and Thelephora pseudoversatilis, two new species collected in the same localities of deciduous and sub-perennial tropical forests of Jalisco, Mexico. Basidiomes of both species are brownish gray to violet brown with clavarioid-mesopodal, sub-resupinate or completely resupinate growth forms. In turn, phylogenetic analyses using nrDNA ITS sequences showed that these species are not closed related, nevertheless they are part of a well-supported clade conformed by several species of Thelephora, Tomentella and some undescribed Thelephorales. Morphological segregation of these species was attained by analyzing spore and hyphae characters using a wide sample. Significant statistical differences between the new species were observed regarding spore size, spine size and context hyphae width. This work exemplifies the relevance of integrating both morphological and molecular data, as well of the use of an appropriate sample size in order to discriminate among morphological cryptic species.

Cytology and molecular phylogenetics of Monoblepharidomycetes provide evidence for multiple independent origins of the hyphal habit in the Fungi.

The evolution of filamentous hyphae underlies an astounding diversity of fungal form and function. We studied the cellular structure and evolutionary origins of the filamentous form in the Monoblepharidomycetes (Chytridiomycota), an early-diverging fungal lineage that displays an exceptional range of body types, from crescent-shaped single cells to sprawling hyphae. To do so, we combined light and transmission electron microscopic analyses of hyphal cytoplasm with molecular phylogenetic reconstructions. Hyphae of Monoblepharidomycetes lack a complex aggregation of secretory vesicles at the hyphal apex (i.e. Spitzenkörper), have centrosomes as primary microtubule organizing centers and have stacked Golgi cisternae instead of tubular/fenestrated Golgi equivalents. The cytoplasmic distribution of actin in Monoblepharidomycetes is comparable to the arrangement observed previously in other filamentous fungi. To discern the origins of Monoblepharidomycetes hyphae, we inferred a phylogeny of the fungi based on 18S and 28S ribosomal DNA sequence data with maximum likelihood and Bayesian inference methods. We focused sampling on Monoblepharidomycetes to infer intergeneric relationships within the class and determined 78 new sequences. Analyses showed class Monoblepharidomycetes to be monophyletic and nested within Chytridiomycota. Hyphal Monoblepharidomycetes formed a clade sister to the genera without hyphae, Harpochytrium and Oedogoniomyces. A likelihood ancestral state reconstruction indicated that hyphae arose independently within the Monoblepharidomycetes lineage and in at least two other lineages. Cytological differences among monoblepharidalean and other fungal hyphae are consistent with these convergent origins.

Cantharellaceae of Guyana II: new species of Craterellus, new South American distribution records for Cantharellus guyanensis and Craterellus excelsus, and a key to the Neotropical taxa.

Craterellus olivaceoluteus sp. nov. and Craterellus cinereofimbriatus sp. nov. are described as new to science. These fungi were collected from Guyana in association with ectomycorrhizal host trees in the genera Dicymbe (Fabaceae subfam. Caesalpinioideae) and Pakaraimaea (Dipterocarpaceae). Cantharellus guyanensis Mont., originally described from French Guiana, is redescribed from recent collections from Guyana, with additional range extensions for the species provided based on material examined from French Guiana, Venezuela, and north central, northeastern and southern Brazil, circumscribing nearly the entire Guiana Shield region and beyond. A new distribution record from French Guiana is provided for Craterellus excelsus T.W. Henkel & Aime. Macromorphological, micromorphological and habitat data are provided for the new species and C. guyanensis as well as DNA sequence data from the nuclear ribosomal regions of the internal transcribed spacer (ITS) and 28S large subunit (LSU); additional sequence data is provided for C. guyanensis and C. excelsus specimens collected outside Guyana. The relationships of these taxa within the Cantharellaceae were evaluated with phylogenetic analyses of ITS and LSU sequence data. This work brings the total number of Cantharellaceae species known from Guyana to eight. A key to the Cantharellus and Craterellus species known from the lowland Neotropics and extralimital montane Central and South America is provided.

[Biological characteristics of an Hog1 MAPK homologous gene FoHog1 knock-out mutant of Fusarium oxysporum f. sp. cubense].

OBJECTIVE: This study was aimed to obtain a mitogen-activated protein kinase (MAPK) gene namely FoHog1 from Fusarium oxysporum f. sp. cubense and to verify its function. METHODS: We amplified FoHog1 gene by PCR and RT-PCR methods and analyzed it through bioinformatics method. PEG-mediated protoplast transformation was used to create the deletion mutants of FoHog1 gene. We analyzed different biological characteristics between knock-out strain and wild-type strain. RESULTS: FoHog1 gene encoding a putative protein of 357 amino acids and its genetic relationship with different Fusarium' s protein. Compared with the wild-type strain, FoHog1 deletion mutants have loose hyphae colony, less spores production, lower dry weight of hyphae and more sensitive to temperature, pH and osmotic stress. FoHog1 deletion mutants also have reduced colonization ability compared with the wild-type strain. CONCLUSION: FoHog1 gene participated in mycelial growth, sporulation, catabolism of sodium acetate and ammonium chloride, osmotic stress response and pathogenic process with Fusarium oxysporum f. sp. cubense Race 4.

Primary hepatic aspergillosis following induction chemotherapy for acute leukemia.

Invasive aspergillosis (IA) contributes significantly to the burden of infectious complications in heavily immunosuppressed patients with acute leukemia. The infection is typically acquired via inhalation into the respiratory tract, and the lungs are most commonly involved. However, disseminated disease may occur and reports of isolated extrapulmonary infection suggest the gastrointestinal tract is likely an additional portal of entry for this organism. We describe a case of primary hepatic aspergillosis in a patient with acute myelogenous leukemia. The patient did not respond to medical therapy with antifungals and ultimately required surgical exploration and drainage. IA should be considered in an immunosuppressed patient with hepatic abscesses and may require a combined surgical and medical approach to therapy.

Vegetative compatibility and anastomosis formation within and among individual germlings of tropical isolates of arbuscular mycorrhizal fungi (Glomeromycota).

Hyphal anastomoses which play a key role in the formation of interconnected mycorrhizal networks and in genetic exchange among compatible individuals have been studied in a limited number of species and isolates of arbuscular mycorrhizal fungi (AMF), mainly in symbiotic mycelium. In this work, the occurrence and frequency of anastomosis between hyphae of the same and different germlings were assessed in tropical isolates belonging to Acaulospora, Claroideoglomus, Gigaspora, Glomus, Rhizophagus and Scutellospora. Germlings belonging to Acaulospora, Claroideoglomus, Glomus and Rhizophagus formed perfect hyphal fusions, with frequencies ranging from 9.29 ± 3.01 to 79.84 ± 4.39 % within the same germling and from 14.02 ± 7.36 to 91.41 ± 3.92 % between different germlings. Rare fusions, occurring within the same hypha, were detected in Gigaspora species, and no anastomoses were observed in Scutellospora species. The consistent detection of nuclei in perfect fusions suggests that nuclear migration is active both within and between germlings. Present data on anastomosis formation, nuclear migration and germling viability in tropical isolates of AMF widen our knowledge on the extensive and consistent occurrence of successful hyphal fusions in this group of beneficial symbionts. The ability to anastomose and establish protoplasm flow, fundamental for the maintenance of physiological and genetic continuity, may produce important fitness consequences for the obligately biotrophic AMF.

A new Cortinarius of section Calochroi (Basidiomycota, Agaricomycetes) from Mediterranean Quercus woodlands (Italy).

A new species of Cortinarius, C. flavoaurantians sp. nov., is described from Italian Quercus woods based on both morphological and ITS rDNA data. This taxon is characterized by a yellowish pileus and cortina, a white universal veil and a pileipellis that reacts yellow-orange with KOH. Illustrations of the key micromorphological features and fresh basidiomata in situ are provided. Closely related species are discussed.

3-D imaging of temporal and spatial development of Puccinia striiformis haustoria in wheat.

Differentiation of haustoria on primary infection hyphae of the fungal pathogen Puccinia striiformis was studied in wheat seedlings with two-photon microscopy in combination with a classical staining technique. Our results showed a significant increase in the average haustorium size 22, 44, 68, 92 and 116 h after inoculation (hai). After 116 hai no significant change was observed until 336 hai. Haustorium morphology also changed significantly during the time of infection. Initially small spherical haustoria were seen, but as they grew the haustoria gradually became apically branched. At 22 hai all observed haustoria were spherical, but at 44 hai most haustoria had an irregular structure, and at 92 hai all observed haustoria appeared branched. Along with the changes of the haustorial body the haustorial neck changed from narrow and slender to having an expanded appearance with a rough and invaginated structure. The structural changes were similar in two susceptible wheat varieties, 514W and Cartago, although the mean haustorium size was larger in 514W than in Cartago at all intervals.

Ectomycorrhizal association of three Lactarius species with Carpinus and Quercus trees in a Mexican montane cloud forest.

Ectomycorrhizal (EM) fungi are being monitored in the Santuario del Bosque de Niebla in the central region of Veracruz (eastern Mexico). Based on the comparison of DNA sequences (ITS rDNA) of spatiotemporally co-occurring basidiomes and EM root tips, we discovered the EM symbiosis of Lactarius indigo, L. areolatus and L. strigosipes with Carpinus caroliniana, Quercus xalapensis and Quercus spp. The host of the EM tips was identified by comparison of the large subunit of the ribulose-bisphosphate carboxylase gene (rbcL). Descriptions coupled with photographs of ectomycorrhizas and basidiomes are presented.

Three new ascomycetes from freshwater in China.

Three new freshwater ascomycetes, Diaporthe aquatica sp. nov. (Diaporthaceae), Ophioceras aquaticus sp. nov. (Magnaporthaceae) and Togninia aquatica sp. nov. (Togniniaceae), are described and illustrated based on morphological and molecular data (ITS, 18S, 28S rDNA sequences). Diaporthe aquatica is characterized by globose to subglobose, black ascomata with long necks, broadly cylindrical to obclavate asci, and small, ellipsoidal to fusiform, one-septate, hyaline ascospores; it is unusual among Diaporthe species in the fact that it lacks a stroma and has freshwater habitat. Ophioceras aquaticus is characterized by globose ascomata with a long beak, cylindrical, eight-spored asci with J- subapical rings and 3-5-septate filiform ascospores with slightly acute ends. Togninia aquatica is characterized by globose ascomata with long necks, clavate and truncate asci clustered on distinct ascogenous hyphae, and small, reniform, hyaline ascospores. Differences among the new taxa and similar species are discussed.

Hydroxyurea enhances post-fusion hyphal extension during sexual development in C. neoformans var. grubii.

Mating and sexual development in C. neoformans var. grubii strains of the H99 background is often less robust than that laboratory generated isogenic C. neoformans var. neoformans strains in the JEC21 background. In Candida albicans and Saccharomyces serevisiae, slowing of DNA synthesis and engagement of the replication stress response, such as that caused by treatment with hydroxyurea (HU), induces filamentation and pseudohyphal growth, respectively. In this study, we investigated the effect of HU treatment on C. neoformans var. grubii morphogenesis. Treatment with HU did not induce filamentation of yeast cells either in liquid culture or on solid YPD or V8 agar. In the presence of the opposite mating partner, we observed early emergence of hyphae in the presence of HU. Semi-quantitative analysis of fusion using marked strains demonstrated that no significant enhancement of fusion in the presence of HU. Transfer of fusion colonies from crosses performed in the absence of HU to V8 + HU revealed enhanced hyphal growth in the presence of HU. Analysis of expression of the target of HU, ribonucleotide reductase, revealed that a phylogenetically divergent catalytic subunit is replication stress responsive in C. neoformans. These results suggest that induction of replication stress promotes post-fusion hyphal growth of C. neoformans var. grubii strains in the H99 background.

Steccherinum tenuissimum and S. xanthum spp. nov. (Polyporales, Basidiomycota): New species from China.

Two new wood-inhabiting fungal species, Steccherinum tenuissimum and S. xanthum spp. nov. are described based on a combination of morphological features and molecular evidence. Steccherinum tenuissimum is characterized by an annual growth habit, resupinate basidiomata with an odontioid hymenial surface, a dimitic hyphal system with clamped generative hyphae, strongly encrusted cystidia and basidiospores measuring 3-5 × 2-3.5 µm. Steccherinum xanthum is characterized by odontioid basidiomata and a monomitic hyphal system with generative hyphae bearing clamp connections and covering by crystals, colourless, thin-walled, smooth, IKI-, CB-and has basidiospores measuring 2.7-5.5 × 1.8-4.0 µm. Sequences of the ITS and nLSU nrRNA gene regions of the studied samples were generated, and phylogenetic analyses were performed with maximum likelihood, maximum parsimony and Bayesian inference methods. The phylogenetic analyses based on molecular data of ITS + nLSU sequences showed that two new Steccherinum species felled into the residual polyporoid clade. Further investigation was obtained for more representative taxa in Steccherinum based on ITS + nLSU sequences, which demonstrated that S. tenuissimum and S. xanthum were sister to S. robustius with high support (100% BP, 100% BS and 1.00 BPP).