Halo- and psychrotolerant Geomyces fungi from arctic cryopegs and marine deposits

Comparative characterization of Geomyces isolates was performed. The isolates were obtained from Arctic cryopegs and the surrounding ancient marine deposits, from nonsaline permafrost soils, and from temperate environments. Microbiological (cultural and morphological) and molecular criteria were used to confirm the identification of the isolates as Geomyces pannorum. The isolates from cryopegs and surrounding marine deposits were shown to differ from those obtained from nonsaline soils and temperate environments in their ability to grow at negative temperatures (-2 degrees C) under increased salt concentration (10%). The results are discussed in relation to the possible inheritance of the adaptive characteristics acquired in specific environments.     

Microbiological analysis of cryopegs from the Varandei Peninsula,Barents Sea

The paper deals with the microbiological characterization of water-saturated horizons in permafrost soils (cryopegs) found on the Varandei Peninsula (Barents Sea coast), 4–20 m deep. The total quantity of bacteria in the water of cryopegs was 3.5 × 10⁸ cells/ml. The population of cultivated aerobic heterotrophic bacteria was 3–4 × 10⁷ cells/ml and the number of anaerobic heterotrophic bacteria varied from 10² to 10⁵ cells/ml depending on cultivation temperature and salinity. Sulfate-reducing bacteria and methanogenic archaea were found as hundreds and tens of cells per ml of water, respectively. A pure culture of a sulfate-reducing strain B15 was isolated from borehole 21 and characterized. Phylogenetic analysis has shown that the new bacterium is a member of the genus Desulfovibrio with Desulfovibrio mexicanus as its closest relative (96.5% similarity). However, the significant phenotypic differences suggest that strain B15 is a new species of sulfate-reducing bacteria.     

Phenotypic and genetic characterization of rhizobia associated with alfalfa in the Hokkaido and Ishigaki regions of Japan

Twenty five rhizobial isolates were obtained from root nodules of Medicago sativa inoculated with soil samples collected from the Sapporo region and Ishigaki Island in Japan. To study their diversity and characterize them in relation to the climatic conditions of their soils of origin, a polyphasic approach analyzing stress tolerance, symbiotic and genetic properties was used. Stress tolerance assays revealed marked variations in salinity, pH and temperature tolerance. Isolates originating from a sub-tropical climate in alkaline soil (Ishigaki Island) tolerated high temperature, salinity and pH levels. Moreover, isolates recovered from a temperate climate in acidic soil (Sapporo) were sensitive to high temperature and salinity, and tolerated acidic pH. Phylogenetic analysis of conserved 16S rRNA and recA genes, and symbiotic nodA and nifDK revealed 25 isolates to be closely related to Ensifer meliloti. Furthermore, the branch patterns of phylogenetic trees constructed from different genes revealed the existence of at least two E. meliloti types in the soils studied. These results may be relevant to programs directed towards improving crop productivity through biofertilization with locally adapted and genetically defined strains.     

Characteristics of Woodland Rhizobial Populations from Surface- and Deep-Soil Environments of the Sonoran Desert

A collection of 74 rhizobial isolates recovered from nodules of the desert woody legumes Prosopis glandulosa, Psorothamnus spinosus, and Acacia constricta were characterized by using 61 nutritional and biochemical tests. We compared isolates from A. constricta and Prosopis glandulosa and tested the hypothesis that the rhizobia from a deep-phreatic rooting zone of a Prosopis woodland in the Sonoran Desert of southern California were phenetically distinct from rhizobia from surface soils. Cluster analysis identified four major homogeneous groups. The first phenon contained slow-growing (SG) Prosopis rhizobia from surface and deep-phreatic-soil environments. These isolates grew poorly on most of the media used in the study, probably because of their requirement for a high medium pH. The second group of isolates primarily contained SG Prosopis rhizobia from the deep-phreatic rooting environment and included two fast-growing (FG) Psorothamnus rhizobia. These isolates were nutritionally versatile and grew over a broad pH range. The third major phenon was composed mainly of FG Prosopis rhizobia from surface and dry subsurface soils. While these isolates used a restricted range of carbohydrates (including sucrose) as sole carbon sources, they showed better growth on a range of organic acids as sole carbon sources and amino acids as sole carbon and nitrogen sources than did other isolates in the study. They grew better at 36°C than at 26°C. The FG Acacia rhizobia from surface-soil environments formed a final major phenon that was distinct from the Prosopis isolates. They produced very high absorbance readings on all of the carbohydrates tested except sucrose, grew poorly on many of the other substrates tested, and preferred a 36 to a 26°C incubation temperature. The surface populations of Prosopis rhizobia required a higher pH for growth and, under the conditions used in this study, were less tolerant of low solute potential and high growth temperature than were phreatic-soil isolates. SG Prosopis rhizobia from phreatic and surface soils were physiologically distinct, suggesting adaptation to their respective soil environments.     

Numerically Dominant Denitrifying Bacteria from World Soils

Nineteen soils, three freshwater lake sediments, and oxidized poultry manure were examined to determine the dominant denitrifier populations. The samples, most shown or expected to support active denitrification, were from eight countries and included rice paddy, temperate agricultural, rain forest, organic, and waste-treated soils. Over 1,500 organisms that could grow anaerobically on nitrate agar were isolated. After purification, 146 denitrifiers were obtained, as verified by production of N₂ from NO₃^-. These isolates were characterized by 52 properties appropriate for the Pseudomonas-Alcaligenes group. Numerical taxonomic procedures were used to group the isolates and compare them with nine known denitrifier species. The major group isolated was representative of Pseudonomas fluorescens biotype II. The second most prevalent group was representative of Alcaligenes. Other Pseudomonas species as well as members of the genus Flavobacterium, the latter previously not known to denitrify, also were identified. One-third of the isolates could not utilize glucose or other carbohydrates as sole carbon sources. Significantly, none of the numerically dominant denitrifiers we isolated resembled the most studied species: Pseudomonas denitrificans, Pseudomonas perfectomarinus, and Paracoccus denitrificans. Denitrification appears to be a property of a very diverse group of gram-negative, motile bacteria, as shown by the large number (22.6%) of ungrouped organisms. The diversity of denitrifiers from a given sample was usually high, with at least two groups present. Denitrifiers, nitrite accumulators, and organisms capable of anaerobic growth were present in the ratio of 0.20±0.23:0.81±0.23:1. There were few correlations between their numbers and the sample characteristics measured. However, the temperatures at which isolates could grow were significantly related to the temperatures of the environments from which they were isolated. Regression analysis revealed few relationships between physical parameters and bacterial types, save for the anaerobe numbers, in which 94% of the variance could be accounted for.     

Review of taxonomy,geographic distribution,and paleoenvironments of Azhdarchidae (Pterosauria)

The taxonomy, geographic distribution, and paleoenvironmental context of azhdarchid pterosaurs are reviewed. All purported pteranodontid, tapejarid, and azhdarchid specimens from the Cenomanian Kem Kem beds of Morocco are referred to a single azhdarchid taxon, Alanqa saharica. The four proposed autapomorphies of Eurazhdarcho langendorfensis from the lower Maastrichtian Sebeş Formation of Romania are based on misinterpretations of material and this taxon is likely a subjective junior synonym of Hatzegopteryx thambema. Among 54 currently reported azhdarchid occurrences (51 skeletal remains and 3 tracks) 13% are from lacustrine deposits, 17% from fluvial plain deposits, 17% from coastal plain deposits, 18% from estuarine and lagoonal deposits, and 35% from costal marine deposits. Azhdarchids likely inhabited a variety of environments, but were abundant near large lakes and rivers and most common in nearshore marine paleoenvironments.     

Vampires in the oceans: predatory cercozoan amoebae in marine habitats

Vampire amoebae (vampyrellids) are predators of algae, fungi, protozoa and small metazoans known primarily from soils and in freshwater habitats. They are among the very few heterotrophic naked, filose and reticulose protists that have received some attention from a morphological and ecological point of view over the last few decades, because of the peculiar mode of feeding of known species. Yet, the true extent of their biodiversity remains largely unknown. Here we use a complementary approach of culturing and sequence database mining to address this issue, focusing our efforts on marine environments, where vampyrellids are very poorly known. We present 10 new vampyrellid isolates, 8 from marine or brackish sediments, and 2 from soil or freshwater sediment. Two of the former correspond to the genera Thalassomyxa Grell and Penardia Cash for which sequence data were previously unavailable. Small-subunit ribosomal DNA analysis confirms they are all related to previously sequenced vampyrellids. An exhaustive screening of the NCBI GenBank database and of 454 sequence data generated by the European BioMarKs consortium revealed hundreds of distinct environmental vampyrellid sequences. We show that vampyrellids are much more diverse than previously thought, especially in marine habitats. Our new isolates, which cover almost the full phylogenetic range of vampyrellid sequences revealed in this study, offer a rare opportunity to integrate data from environmental DNA surveys with phenotypic information. However, the very large genetic diversity we highlight within vampyrellids (especially in marine sediments and soils) contrasts with the paradoxically low morphological distinctiveness we observed across our isolates.     

Strain Variation in Mycobacteriummarinum Fish Isolates

A molecular characterization of two Mycobacterium marinum genes, 16S rRNA and hsp65, was carried out with a total of 21 isolates from various species of fish from both marine and freshwater environments of Israel, Europe, and the Far East. The nucleotide sequences of both genes revealed that all M. marinum isolates from fish in Israel belonged to two different strains, one infecting marine (cultured and wild) fish and the other infecting freshwater (cultured) fish. A restriction enzyme map based on the nucleotide sequences of both genes confirmed the divergence of the Israeli marine isolates from the freshwater isolates and differentiated the Israeli isolates from the foreign isolates, with the exception of one of three Greek isolates from marine fish which was identical to the Israeli marine isolates. The second isolate from Greece exhibited a single base alteration in the 16S rRNA sequence, whereas the third isolate was most likely a new Mycobacterium species. Isolates from Denmark and Thailand shared high sequence homology to complete identity with reference strain ATCC 927. Combined analysis of the two gene sequences increased the detection of intraspecific variations and was thus of importance in studying the taxonomy and epidemiology of this aquatic pathogen. Whether the Israeli M. marinum strain infecting marine fish is endemic to the Red Sea and found extremely susceptible hosts in the exotic species imported for aquaculture or rather was accidentally introduced with occasional imports of fingerlings from the Mediterranean Sea could not be determined.     

Isolation and characterization ofVibrio vulnificus inhabiting the marine environment of the southwestern area of Taiwan

Vibrio vulnificus, a marine bacterium, is of concern in Taiwan because it causes wound infections and sepsis with a high mortality rate every year. To examine forV. vulnificus, 13 samples of seawater or oysters were collected from nine sites in Yunlin, Chiayi, and Tainan. Seventy-seven strains ofV. vulnificus were isolated from 11 samples. Among these environmental isolates, 72 (91%) were indole-positive, a characteristic of biotype 1. The remaining five strains although indole-negative, a characteristic previously found exclusively in biotype 2 strains, were all ornithine decarboxylase- and mannitol-positive, which has never been reported for biotype 2 strains. Based on the overall biochemical reactions obtained using a commercial identification system, these indole-negative strains appeared to be more like biotype 1. Fifty-seven ribotypes were identified among these isolates, indicating the great genetic divergence in this species. Of the 30 environmental isolates tested, 17 (56.7%) exhibited virulence comparable to the clinical isolates in the mouse, implying that a high proportion of theV. vulnificus strains in the marine environments might be pathogenic to humans.     

Characterization of feather-degrading bacteria from Brazilian soils

Studies on keratinolytic microorganisms have been mainly related to their biotechnological applications and association with animal pathologies. However, these organisms have an ecological relevance to recycling keratinous residues in nature. This work aimed to select and identify new culturable feather-degrading bacteria isolated from soils of Brazilian Amazon forest and Atlantic forest. Bacteria that were isolated from temperate soils and bacteria from Amazonian basin soil were tested for their capability to grow on feather meal agar (FMA). Proteolytic bacteria were tested for feather degradation and were further identified according to their morphological and biochemical characteristics. Also, molecular identification based on 16S rDNA gene sequencing was carried out. A total of 24 proteolytic and 20 feather-degrading isolates were selected; Most of the isolates were from the Bacillus genus (division Firmicutes), but one Aeromonas, two Serratia (??-Proteobacteria), and one Chryseobacterium (Cytophaga-Flavobacterium group).     

Diversity of aerobic and facultative alkalitolerant and halotolerant endospore formers in soil from the Alvord Basin,Oregon

Many proteins produced by Bacillus species isolated from extreme environments have been utilized for industrial purposes, as these extreme environments often promote evolution of unique protein properties. The Borax Lake area is unusual due to its geothermal activity, elevated pH, and high arsenic and salt concentrations in its soils. Soils from this region are likely to harbor alkalitolerant, halotolerant, endospore-forming strains that may be of potential ecological and/or commercial interest. The objectives of this study were to develop new PCR primers that could target Bacillus or closely related 16S rRNA genes, to characterize the diversity of alkalitolerant, halotolerant, endospore-forming organisms in the soils surrounding Borax Lake, and to identify novel organisms that may ultimately provide new enzymes for applied use. A three-pronged approach was used to identify such bacteria in soil samples. Organisms were isolated using two different techniques. Finally, metagenomic DNA from soil samples was subjected to 16S rRNA gene amplification using the newly designed primers. Assays were performed to characterize the halotolerance and alkalitolerance of isolates.     

Nitrogen mineralization rates in salinevs. salt-amended soils

Studies were conducted to compare N mineralization rates in salt-amended nonsaline soils to naturally-occurring saline soils. NaCl, CaCl₂, and Na₂SO₄ were added to nonsaline soils at rates that produced electrical conductivities of the saturation extracts (EC_e) of 5, 10, 15, and 20 dS m⁻¹. Saline soils with similar properties were leached to the same EC_c levels. N mineralization in the Chino soil was inhibited by salt addition, particularly with sodium and calcium chlorides. In the Domino soil there was some inhibition of N mineralization with the chloride salts, but enhancement with Na₂SO₄ was observed. Nitrification in both soils was more sensitive to salt addition than ammonification. N mineralization occurred more slowly in both leached saline soils compared to the salt-amended soils. Leached saline soils often accumulated greater amounts of inorganic N compared to their native saline counterparts, particularly with the 5 dS m⁻¹ Chino soil (native, 44 dS m⁻¹) and with the 5, 10, 15 and 20 dS m⁻¹ Domino soils (native, 32 dS m⁻¹). Kinetic parameters were estimated by the linear least squares (LLS) and the nonlinear least squares (NLLS) methods. Generally, the LLS transformation estimated greater values of potentially mineralizable N (N_o) and lower rate constants (k). With the NLLS equation, N_o values for the leached saline soils were usually lower, and k values usually higher than in the salt-amended soils. The nonsaline controls generally had the highest N_o and lowest k estimates. Average LLS rate constants for the salt-amended and leached saline soils were 0.055 and 0.083 for the Chino, and 0.104 and 0.137 week⁻¹, respectively, for the Domino soils. With the NLLS equation, average k values for the salt-amended and leached saline soils were 0.087 and 0.089 for the Chino, and 0.181 and 0.387 week⁻¹, respectively, for the Domino soils. These results suggest that N mineralization rates obtained in salt-amended nonsaline soils may not be representative of those in naturally-occurring saline soils.     

Sphingomonads from marine environments

Sphingomonas species play an important role in the ecology of a range of marine habitats. Isolates and 16S-rRNA clones have been obtained from corals, natural and artificial sources of marine hydrocarbons and eutrophic and oligotrophic waters, and have been isolated as hosts for marine phages. In addition they are found in oceans spanning temperature ranges from polar to temperate waters. While less is known about marine sphingomonads in comparison to their terrestrial counterparts, their importance in microbial ecology is evident. This is illustrated by, for example, the numerical dominance of strain RB2256 in oligotrophic waters. Furthermore, the known marine sphingomonads represent a phylogenetic cross-section of the Sphingomonas genus. This review focuses on our present knowledge of cultured isolates and 16S-rDNA clones from marine environments. Received 01 May 1999/ Accepted in revised form 13 July 1999     

Bacillus Endospores Isolated from Granite: Close Molecular Relationships to Globally Distributed Bacillus spp. from Endolithic and Extreme Environments

As part of an ongoing effort to catalog spore-forming bacterial populations in environments conducive to interplanetary transfer by natural impacts or by human spaceflight activities, spores of Bacillus spp. were isolated and characterized from the interior of near-subsurface granite rock collected from the Santa Catalina Mountains, AZ. Granite was found to contain ~500 cultivable Bacillus spores and ~10⁴ total cultivable bacteria per gram. Many of the Bacillus isolates produced a previously unreported diffusible blue fluorescent compound. Two strains of eight tested exhibited increased spore UV resistance relative to a standard Bacillus subtilis UV biodosimetry strain. Fifty-six isolates were identified by repetitive extragenic palindromic PCR (rep-PCR) and 16S rRNA gene analysis as most closely related to B. megaterium (15 isolates), B. simplex (23 isolates), B. drentensis (6 isolates), B. niacini (7 isolates), and, likely, a new species related to B. barbaricus (5 isolates). Granite isolates were very closely related to a limited number of Bacillus spp. previously found to inhabit (i) globally distributed endolithic sites such as biodeteriorated murals, stone tombs, underground caverns, and rock concretions and (ii) extreme environments such as Antarctic soils, deep sea floor sediments, and spacecraft assembly facilities. Thus, it appears that the occurrence of Bacillus spp. in endolithic or extreme environments is not accidental but that these environments create unique niches excluding most Bacillus spp. but to which a limited number of Bacillus spp. are specifically adapted.     

Foraminiferal biostratigraphy of the lower Miocene Hamzian and Arashtanab sections (NW Iran), northern margin of the Tethyan Seaway

Lower Miocene strata exposed in the Khoy-Bostan Abad area (NW Iran) document marine platform environments. Paleontological and lithological characteristics of these deposits were investigated to develop detailed bio- and lithostratigraphic frameworks. The shallow marine units, composed of bioclastic carbonates in Bostan Abad and mixed siliciclastic carbonates in Khoy area, were deposited under different local tectonic regimes. Relatively diverse assemblages of benthic foraminifera from these shallow marine units were studied to establish a high resolution biostratigraphy in the context of the standard shallow benthic zonation of Western Tethys. The co-occurrence of Miogypsina intermedia Drooger and Borelis melo curdica (Reichel) characterizes Zone SBZ25, indicating a Burdigalian age as also indicated by planktonic foraminifera.     

Tropical oomycetes in the German Bight – Climate warming or overlooked diversity?

Oomycetes have colonised both terrestrial and marine habitats, and leaf litter decomposing oomycetes have been found in coastal and estuarine environments, yet their ecological relevance remains unclear. In tropical and subtropical ecosystems, Halophytophthora species were reported from mangroves and salt marshes, and recently, the genus Salisapilia was described based on oomycetes recovered from leaf litter in subtropical Georgia, USA. In cool temperate regions, only Pythium and Phytophthora species have so far been reported from coastal environments. Here we report the presence of two species of Halophytophthora in the German Bight, north of Hamburg. The presence of a group of oomycetes, previously reported only from the subtropics and tropics, in the cool temperate German Bight raises the question of whether these were previously overlooked, introduced with ballast water or have migrated there as a result of climate change. Temperature preference of the strains hints at the first hypothesis.     

Genetics and Morphology Characterize the Dinoflagellate Symbiodinium voratum,n. sp., (Dinophyceae) as the Sole Representative of Symbiodinium Clade E

Dinoflagellates in the genus Symbiodinium are ubiquitous in shallow marine habitats where they commonly exist in symbiosis with cnidarians. Attempts to culture them often retrieve isolates that may not be symbiotic, but instead exist as free‐living species. In particular, cultures of Symbiodinium clade E obtained from temperate environments were recently shown to feed phagotrophically on bacteria and microalgae. Genetic, behavioral, and morphological evidence indicate that strains of clade E obtained from the northwestern, southwestern, and northeastern temperate Pacific Ocean as well as the Mediterranean Sea constitute a single species: Symbiodinium voratum n. sp. Chloroplast ribosomal 23S and mitochondrial cytochrome b nucleotide sequences were the same for all isolates. The D1/D2 domains of nuclear ribosomal DNA were identical among Western Pacific strains, but single nucleotide substitutions differentiated isolates from California (USA) and Spain. Phylogenetic analyses demonstrated that S. voratum is well‐separated evolutionarily from other Symbiodinium spp. The motile, or mastigote, cells from different cultures were morphologically similar when observed using light, scanning, and transmission electron microscopy; and the first complete Kofoidian plate formula for a Symbiodinium sp. was characterized. As the largest of known Symbiodinium spp., the average coccoid cell diameters measured among cultured isolates ranged between 12.2 (± 0.2 SE) and 13.3 (± 0.2 SE) μm. Unique among species in the genus, a high proportion (approximately 10–20%) of cells remain motile in culture during the dark cycle. Although S. voratum occurs on surfaces of various substrates and is potentially common in the plankton of coastal areas, it may be incapable of forming stable mutualistic symbioses.     

Bacilli community of saline–alkaline soils from the Ararat Plain (Armenia) assessed by molecular and culture-based methods

The bacterial community composition in the A horizon of a natural saline–alkaline soil located in Ararat Plain (Armenia) was studied using molecular and culture-based methods The sequence analysis of a 16S rRNA gene clone library and denaturing gradient gel electrophoresis (DGGE) profiles indicated dominance of Firmicutes populations. The majority of the sequences of the bacterial 16S rRNA gene library were close relatives of representatives belonging to the genera Halobacillus (41.2%), Piscibacillus (23.5%), Bacillus (23.5%) and Virgibacillus (11.8%). Eight novel moderately halophilic bacilli isolates were successfully obtained from the enriched cultures of the saline–alkaline soil samples. 16S rRNA gene sequence analyses of isolates revealed their affiliation (97.7–99.7% similarity) to representatives of the genera Bacillus, Piscibacillus and Halobacillus. All isolates were able to tolerate high concentrations of NaCl and highly alkaline conditions. This is the first study combining cultivation-independent and -dependent approaches to reveal the bacterial diversity of the saline–alkaline soils of Ararat Plain and it suggested an important role of bacilli as key microbes in biogeochemical cycles of these environments.     

Palaeoenvironmental distribution of the Rhaetian dinoflagellate cysts Dapcodinium priscum EVITT, 1961, emend. Below, 1987 and Rhaetogonyaulax rhaetica (SARJEANT) LOEBLICH and LOEBLICH, 1976, emend. Harland et al., 1975, emend. Below, 1987

In almost all Rhaetian marine assemblages of the Tethyan Realm, two dinoflagellate cyst species, Dapcodinium priscum EVITT, 1961, emend. Below, 1987, and Rhaetogonyaulax rhaetica (SARJEANT) LOEBLICH and LOEBLICH, 1976, emend. Harland et al., 1975, emend. Below 1987 are dominant. Thus, when one is relatively frequent the other scarce and vice versa. This paper considers the inverse relationship of the two species in 235 rock samples taken from Rhaetian strata well exposed along the western margin of the Mesozoic intracratonic basin of south-eastern France. The palynological variables recorded from the samples (seven types of phytoclasts and three marine taxa: D. priscum, R. rhaetica and micrhystridids) were submitted to principal components analysis, which shows that D. priscum occurs in deposits that reflect nearshore and restricted marine environments. R. rhaetica is indicative of more marine environments at greater water depth, as indicated by the relatively high proportions of buoyant needle-like phytoclasts. These observations suggest that D. priscum is the cyst of an opportunistic, euryhaline dinoflagellate taxon that occupied various ecological niches while R. rhaetica inhabited more normal open marine environments. The palaeoenvironmental distribution seems to have been influenced by the energy level of the water column. D. priscum is found in deposits that denote high to low energy while R. rhaetica is found in low energy sediments. Micrhystridid occurrences are similar to those of D. priscum; they are uncommon when the abundance of R. rhaetica increases.     

Bradyrhizobium Populations Occur in Deep Soil under the Leguminous Tree Acacia albida

Soil cores were drilled under the leguminous tree Acacia albida growing in two different ecoclimatic zones of West Africa: the Sahelian area (100 to 500 mm of annual rainfall) and the Sudano-Guinean area (1,000 to 1,500 mm of annual rainfall). Soil samples were collected at different depths from the surface down to the water table level and analyzed for the presence of rhizobia able to nodulate A. albida. In both areas, population densities of rhizobia were substantially greater near the water table than near the surface. In the Sahelian area, rhizobia were present as deep as 34 m at a concentration of 1.3 × 10³/g of soil. In the Sudano-Guinean area, population densities at 0.5 to 4.5 m depth were higher than in the Sahelian area and, at several depths, comparable to that of temperate soils supporting legume crops (10⁴ rhizobia per g of soil). Surface and deep soil isolates from all four sites were found to be slow-growing rhizobia (Bradyrhizobium sp.). The proportion of effective isolates was almost the same within surface and deep soils.