Soil mycoflora from trufflè native areas of Saudi Arabia

Soil fungi of areas in the North-Eastern region of Saudi Arabia where truffles are native were surveyed. Forty-three species of fungi belonging to twenty genera were isolated. Most were recovered from soils underneath or around truffle ascocarps: thirty species from soil under the surface of Tirmania nivea ascocarps, twenty-four from Terfezia boudieri soil and twenty species each from Tirmania pinoyi and Terfezia claveryi soils. Rhizosphere soil of Helianthemum lippi, on the other hand, yielded twenty-four fungal species while only fourteen fungal species were found in soil without vegetation. The total counts of fungi/g soil were highest in soils from the under surface of truffles, followed by rhizosphere soil, with the lowest in soils without vegetation.     

Fungi recovered from root-knot nematodes infecting vegetables under protected cultivation

A survey was conducted in root-knot nematode-infested plastic houses to determine the diversity and frequency of occurrence of fungi associated with the nematode. The relationships between percentage fungal parasitism and physicochemical properties of soil were also investigated. Fifty-nine plastic houses were sampled in southeastern Spain, 42 treated with nematicides and 17 left untreated. Eleven fungal genera and unidentified fungi were isolated from nematode eggs or juveniles. Fungal parasitism occurred more frequently in untreated (82.4%) than treated (50%) soils. The species richness in untreated soils ranged from 0 to 5, the Shannon–Wiener diversity index (a measurement of how many different fungi there are in site taking into account how evenly they are distributed among the site) from 0 to 2.01, and the evenness index from 0.46 to 0.99. In treated soils, species richness ranged from 0 to 4, the Shannon–Wiener diversity index from 0 to 1.61, and the evenness index from 0.81 to 1. Of the sites with nematophagous fungi, Arthrobotrys dactyloides (34%), Cylindrocarpon sp., Neosartoria hiratsukae (17%), and Fusarium solani (14%) were the fungi most frequently found. Physicochemical properties of soil were similar in nematicide treated and untreated soils. Percent fungal parasitism in untreated soils correlated positively with lime, silt and carbonate content of soil.     

Distribution,occurrence and characterization of entomopathogenic fungi in agricultural soil in the Palestinian area

The occurrence of entomopathogenic fungi was investigated in irrigated vegetable fields and citrus orchards soils, over a nine-month period (April-December 1999),using the Galleria bait method (GBM). Entomopathogenic fungi were found to occur in 33.6% of the soil samples studied, with positive samples yielding 70 fungal isolates, belonging to 20 species from 13 genera. Conidiobolus coronatus was the most frequent and abundant entomopathogenic species recovered, comprising 31.4% of the total number of isolates. Soil pH, soil moisture content and the geographical location had minor or no effect on the isolation of entomopathogenic fungi in the fields studied. On the other hand, organic matter content of soil, and vegetation type were found to significantly affect the occurrence of entomopathogenic fungi in soil habitats, with orchard fields yielding larger numbers of isolates than vegetable fields. Using Koch's postulates the pathogenicity of fungal isolates to Galleria larvae was found to range from 16–100% (mortality rate). Isolates of C. coronatus proved to be the most virulent isolates recovered. The effect of media and temperature on mycelial growth rate, conidial production and conidial germination of six entomopathogenic fungal species (C. coronatus, Entomophaga grylli, Erynia castrans, Hirsutella jonesii, Paecilomyces farinosus and Sporodiniella umbellata) was also studied. Mycelial growth rate, spore production and spore germination were significantly affected by media, temperature and isolates. In view of the present results, C. coronatus appears to be a good candidate for pest control in agricultural soils, as it has a wide tolerance to agricultural practices, has frequently been isolated from both vegetable and orchard fields, and is characterized by high mycelial growth rate, conidial production and conidial germination.This revised version was published online in October 2005 with corrections to the Cover Date.     

小蓬竹根际土壤微生物及内生真菌多样性分析

为了解喀斯特典型物种-小蓬竹根际土壤微生物及不同部位内生真菌多样性,采用沿等高线等距离取样法采集小蓬竹根际土壤及健康植株,通过可培养对根际土微生物及内生菌进行分离,利用分子技术对其进行鉴定,根据鉴定结果构建系统发育树,并计算小蓬竹根际土壤微生物和根茎叶内生真菌多样性。结果如下：（1）共从根际土壤、根、茎、叶分离得到139个真菌菌株,隶属于27属,其中根际土壤分离得到34个真菌菌株隶属于12属,根部分离得到的63个内生真菌菌株隶属于17个属,茎部分离得到的14个内生真菌菌株隶属于8个属,叶部分离得到28个内生真菌菌株隶属于9个属;（2）根际土壤共分离得到41株细菌菌株,隶属于7个属26个种,20株放线菌菌株,隶属于1属15种;从Shannon-Wiener多样性指数、均匀度指数、Simpson指数排序来看,真菌主要表现为根 > 根际土壤 > 茎 > 叶,细菌和放线菌多样性均较低。（3）按层次聚类分析可分别将真菌、细菌、放线菌聚为3支。小蓬竹根际土壤、根、茎和叶具有丰富的微生物多样性,不同部位菌群组成存在差异性（P<0.05）,且存在以假单胞菌属、芽孢杆菌属等为优势属的抗盐耐旱菌群,这有助于揭示小蓬竹对喀斯特生境的适应性,以及为微生物-植物群落之间相互关系提供一定基础数据,为后期寻找小蓬竹相关耐性功能菌奠定基础。     

Assessment of soil fungal communities using pyrosequencing

Pyrosequencing, a non-electrophoretic method of DNA sequencing, was used to investigate the extensive fungal community in soils of three islands in the Yellow Sea of Korea, between Korea and China. Pyrosequencing was carried out on amplicons derived from the 5′ region of 18S rDNA. A total of 10,166 reads were obtained, with an average length of 103 bp. The maximum number of fungal phylotypes in soil predicted at 99% similarity was 3,334. The maximum numbers of phylotypes predicted at 97% and 95% similarities were 736 and 286, respectively. Through phylogenetic assignment using BLASTN, a total of 372 tentative taxa were identified. The majority of true fungal sequences recovered in this study belonged to the Ascomycota (182 tentative taxa in 2,708 reads) and Basidiomycota (172 tentative taxa in 6,837 reads). The predominant species of Ascomycota detected have been described as lichen-forming fungi, litter/wood decomposers, plant parasites, endophytes, and saprotrophs: Peltigera neopolydactyla (Lecanoromycetes), Paecilomyces sp. (Sordariomycetes), Phacopsis huuskonenii (Lecanoromycetes), and Raffaelea hennebertii (mitosporicAscomycota). The majority of sequences in the Basidiomycota matched ectomycorrhizal and wood rotting fungi, including species of the Agaricales and Aphyllophorales, respectively. A high number of sequences in the Thelephorales, Boletales, Stereales, Hymenochaetales, and Ceratobasidiomycetes were also detected. By applying high-throughput pyrosequencing, we observed a high diversity of soil fungi and found evidence that pyrosequencing is a reliable technique for investigating fungal communities in soils.     

辽宁碱蓬根际土壤真菌多样性的季节变化及其耐盐性

采用稀释平板法和形态学鉴定法进行了辽宁碱蓬(Suaeda liaotungensis)根际土壤真菌的分离鉴定及季节、盐度对真菌多样性的影响分析。结果表明:春、夏、秋、冬四季辽宁碱蓬根际土壤中的真菌菌落数分别为6410、4180、5730和3340,种类分别为6属13种、9属16种、11属31种、6属12种,共分离鉴定出13属42种真菌;其根际土壤真菌的多样性指数、丰富度指数和均匀度指数,从大到小的次序均为秋夏春冬,3种指数均在秋季达到峰值;多样性指数和丰富度指数差异较大,均匀度指数波动较小,春季和冬季的均匀度指数相近。辽宁碱蓬根际土壤真菌种类明显多于无植被土壤,是无植被土壤的7倍,多样性指数和均匀度指数分别是无植被土壤的4—8倍和1—1.2倍;其根际土壤真菌优势种群包括青霉属(Penicillium)、葡萄穗霉属(Stachybotrys)、枝孢属(Cladosporium)、木霉属(Trichoderma)、曲霉属(Aspergillus)和镰孢属(Fusarium);4个季节的优势菌不尽相同,但均有一个共同的优势菌属青霉属。以Na Cl浓度梯度法制造盐胁迫生境,检测辽宁碱蓬根际土壤真菌对盐胁迫的响应,结果表明:60%左右的菌种能耐受5%以下盐度、15%左右的菌种能耐10%—20%盐度;筛选出了6株高度耐盐菌:细交链孢霉、草酸青霉、产黄青霉、烟曲霉、细极链格孢和赭曲霉,其中最高耐盐菌种赭曲霉可耐20%盐度。创新之处在于以盐度和季节为变量检测辽宁碱蓬根际土壤真菌的种群构成,为其深入研究奠定了理论基础。     

Quantitative and qualitative seasonal changes in the microbial community from the phyllosphere of sugar beet (Beta vulgaris)

Bacteria, yeasts and filamentous fungi colonizing immature, mature and senescing primary leaves of field grown Beta vulgaris (sugar beet) were analysed over a complete growing season. Greatest microbial numbers were detected on senescing primary leaves and these numbers increased over most of the season. The number of colonizers detected on mature leaves was found to be stable over most of the study.Filamentous fungi and yeasts were identified to the genus level and the communities found to have greatest diversity during the summer months. There was no consistent pattern of diversity according to leaf type. Two genera of filamentous fungi, Cladosporium and Alternaria and two yeast genera, Cryptococcus and Sporobolomyces were the most numerous fungal populations isolated. Only 8 filamentous fungi and 3 yeast genera were commonly isolated on PDA (potato dextrose agar).Bacterial strains (1236) were isolated on Tryptic Soy Broth (TSB) agar and identified to species, or in some cases sub-species level, by analysis of their fatty acid methyl ester (FAME) profiles. Isolated bacteria were grouped into 78 named and 37 unnamed species clusters. Greatest number of bacterial species were isolated from young plants and leaves, sampled during the autumn months. Bacterial community diversity was lowest in mid-summer and winter months. Pseudomonas was the most commonly isolated genus and Erwinia herbicola the most common species. P. aureofaciens was the only species isolated from soil that was also isolated from the phyllosphere of B. vulgaris throughout the season.     

Keratinolytic fungi in Egyptian soils

The frequency of occurrence of keratinolytic fungi in seventy soil samples collected from different sites in Upper Egypt and in the coastal area of the Mediterranean and baited with human and animal hair and pigeon feathers was determined.Twenty-one species in addition to an unidentified species, which belong to sixteen genera were collected.Chrysosporium indicum, C. tropicum, C. keratinophilum andMicrosporum gypseum were the most frequent fungal species recovered from baited soils.The eight soil samples collected from the salt marshes of the coastal Mediterranean area were completely free from any keratinolytic fungi, whereas the sixty-two soil samples collected from cultivated soils contributed a species ranging from one to four species.     

Keratinolytic Fungi in Egyptian Soils

The frequency of occurrence of keratinolytic fungi in seventy soil samples, collected from different sites in Upper Egypt and in the coastal area of the Mediterranean, was determined by baiting with human and animal hairs and pigeon feathers. Twenty-one species, in addition to an unidentified species, which belong to sixteen genera were collected. Chrysosporium indicum, C. tropicum, C. keratinophilum, and Microsporum gypseum were the most frequent fungal species recovered from the baited soils. The soil samples collected from the salt marshes of the coastal Mediterranean area were completely free from any keratinolytic fungi, whereas the soil samples collected from cultivated soils contributed species, ranging from one to four species.     

轮作模式对植烟土壤酶活性及真菌群落的影响

种植模式显著影响土壤理化生物学性质,并与作物土传真菌病害的发生密切相关。试验选择云南省具有代表性的红壤,设置烤烟-休闲-玉米(T-B-M)、烤烟-油菜-玉米(T-C-M)、烤烟-油菜-水稻(T-C-R)和烤烟-苕子-水稻(T-V-R)等4种轮作模式,利用化学、酶学分析及454高通量测序技术,研究了土壤养分、酶活性及真菌群落结构,旨在为烤烟的合理轮作提供科学依据。经16a的不同轮作种植后,土壤p H变化于5.6—6.4之间,仍然适合种植水稻、玉米、油菜和烤烟等多种作物。在T-V-R处理的土壤中,烤烟产量、烟叶产值和上中等烟比例最高,有机质比原初提高45.11%,碱解氮、有效磷和微生物量碳氮显著增加,蔗糖酶、脱氢酶、脲酶、磷酸酶和过氧化氢酶活性也显著高于其它轮作模式,说明T-V-R改善了土壤生态环境,促进了微生物繁衍,数量增加,活性增强。土壤真菌的18S r DNA读数依次为13097(T-B-M)、11345(T-C-M)、12939(T-C-R)和13763(T-V-R),分别代表530、378、395和581种(类)的真菌,由子囊菌门、担子菌门、接合菌门、壶菌门和尚待鉴定的真菌等构成,其中尚待鉴定的真菌属种和子囊菌门占绝大部分。在不同轮作处理的土壤中,前15种优势真菌的丰富度变化于29.46%—62.86%之间,优势菌株的相似性极低,说明土壤真菌的种群结构因轮作模式不同而异。T-V-R处理土壤中的真菌多样性指数最高,优势度指数最低,说明T-V-R轮作的土壤适合多种真菌的繁殖生长,种群数量增加。多种真菌共同存在,互相制约,可防止病原真菌过度繁殖,降低作物发生真菌病害几率。从作物产量和产值、土壤有机质、养分和真菌种群结构看,T-V-R优于其它3种轮作模式,值得推广应用。     

Four endoparasitic nematode destroying fungi isolated from Sand Ridge State Forest soil

A survey to determine the endoparasitic nematode destroying fungi located within Sand Ridge State Forest of Illinois was conducted from 1 January to 3 April 1973. A total of seven nematode destroying fungal species were isolated from the collected soil samples. Harposporium helicoides, H. crassum, H. lilliputanum are endoparasitic nematophagous fungi that have been isolated previously from the forest soil. Acrostalagmus gonoides, A. obovatus, Cephalosporium balanoides, and Monacrosporium cionopagum are nematophagous fungal species that had not been isolated previously from Illinois soil. Soil pH's and soil nutrient levels were not important in the isolation frequency of the collected endoparasitic nematode destroying fungi.     

Arbuscular mycorrhizal fungi associated with common pteridophytes in Dujiangyan,southwest China

The colonization and diversity of arbuscular mycorrhizal (AM) fungi associated with common pteridophytes were investigated in Dujiangyan, southwest China. Of the 34 species of ferns from 16 families collected, 31 were colonized by AM fungi. The mean percentage root length colonized was 15%, ranging from 0 to 47%. Nineteen species formed Paris-type and 10 intermediate-type AM. In two ferns, only rare intercellular non-septate hyphae or vesicles were observed in the roots and AM type could not be determined. Of the 40 AM fungal taxa belonging to five genera isolated from rooting-zone soils, 32 belonged to Glomus, five to Acaulospora, one to Archaeospora, one to Entrophospora, and one to Gigaspora. Acaulospora and Glomus were the dominant genera and Glomus versiforme was the most common species. The average AM spore density was 213 per 100 g air-dried soil and the average species richness was 3.7 AM species per soil sample. There was no correlation between spore density and percentage root length colonized by AM fungi.     

Psychrooligotrophic fungi from Arctic soils of Franz Joseph Land

Microfungi isolated from soil samples collected in Franz Joseph Land by means of the soil dilution and soil washing methods were screened for their ability to grow at low temperatures. The oligotrophic ability of the psychrotrophic strains was then determined on silica gel. A total of 40 taxa from 21 genera were isolated. Most isolates were species of Acremonium, Geomyces, Mortierella, Phialophora, Phoma, Thelebolus and sterile fungi. Eutypella scoparia, Hyphozyma variabilis and Ovadendron sulphureo-ochraceum are new records as soil and Arctic fungi. Most fungal isolates were psychrotrophic. The few psychrooligotrophs were species of Geomyces, Phoma, Thelebolus and Mortierella. Overall, our results suggest that the major component of an Arctic soil mycoflora should be active in one or more short growing seasons interspersed with periods of prolonged dormancy. Only a minor component, such as the small group of psychrooligotrophic fungi found in this study, may be expected to show continuous slow growth. Accepted: 22 November 1998     

Nematode-Capturing Hyphomycetes from Soils over Xerophytic Calcareous Rock in Upper Bavaria*

Nematode-capturing hyphomycetes were isolated from soil and plant debris on the south slope of a hill in the Jurassic of Upper Bavaria. The following species were found: Arthrobotrys arthrobotryoides, A. dactyloides, A. oligospora, A. superba, Dactylella astenopaga, Monacrosporium cionopagum, M. psychrophilum. Endozoic fungal parasites of nematodes were not isolated. With one fungal propagule of nematode-destroying fungi in 1.7 g soil, the density is only slightly lower than values given for agricultural soils.     

Keratinophilic fungi isolated from hospital dust and soils of public places at Gulbarga,India

Keratinophilic fungi were isolated from corridor dust of 11 hospitals and soils of 21 public places using a hair baiting technique. A total of 41 species belonging to 24 genera were recovered. Among the dermatophytes and related species, Microsporum gypseum was predominant, followed by Chrysosporium keratinophilum, Trichophyton mentagrophytes, M. nanum and Ch. tropicum. Other species were represented by 32 species belonging to 21 genera. Most of the species isolated are known to be agents of human and animal infection or have been isolated from human and animal lesions. The fungi observed here are discussed in relation to their global distribution.     

Diversity of saprobic microfungi

The data needed to derive an accurate estimate of saprobic microfungi are insufficient, incomplete and contradictory. We therefore address issues that will ultimately reveal whether there are 1.5 million global fungal species, which is the generally accepted working estimate. Our data indicates that large numbers of fungi occur on host families, such as Musaceae, host genera such as Nothofagus and individual host species such as Eucalyptus globulus, and that fungi may be specific or recurrent on different plant groups. Recent studies have shown that fungal numbers on hosts may be larger than originally thought as saprobes are organ-specific/-recurrent and changes in fungal communities occur as substrata decays. Other issues, such as the impact of geography, of methodology and of taxonomy are also addressed. There is evidence that fungi on the same host at different locations also differs; site-specific factors and geographic distance may be more important than host/substrate in shaping fungal assemblages. Methodology impacts on estimates of species diversity with many more taxa observed using indirect isolation protocols as compared to direct isolations from leaves. Our understanding of fungal species numbers in speciose genera is important. In some fungal groups accepted species have been reduced to a few species, while in other groups many cryptic species are being uncovered. While we make a number of generalisations from the studies reported here, this review also highlights some of the limitations mycologists currently have to contend with. A large body of knowledge exists for certain groups of microfungi or for microfungi occurring on certain substrata/hosts. However, it is likely that we are drawing conclusions from data that are somewhat biased toward fungi and host/substrata that are of interest to human endeavours. The discrepancy between the numbers of fungi described from only one economically important genus, Eucalyptus, and all the other members of the Myrtaceae is but one example of this bias. By incorporating the large body of work that is already available and adding appropriate complementary studies, we can accelerate our understanding of microfungal diversity and this will eventually lead us to a realistic estimate of global fungal species numbers.     

Colonization Characteristics and Diversity of Arbuscular Mycorrhizal Fungi in the Rhizosphere of <i>Iris lactea</i> in Songnen Saline-alkaline Grassland

To understand arbuscular mycorrhizal (AM) fungi resources and develop AM fungal species in ornamental plants with saline-alkaline tolerances, Iris lactea, which grows in the Songnen saline-alkaline grassland with a high ornamental value, was selected as the experimental material, and the colonization characteristics of its roots and the AM fungal diversity in its rhizosphere were explored. The results of the observations and calculations of mycorrhizae from ten different samples showed that AM fungi colonized the roots of I. lactea and formed Arum-type mycorrhizal structures. There was a significant correlation between soil spore density and pH value, while the colonization rate showed a fluctuating trend with increasing pH values. The observed colonization intensities were of Levels II (1%–10%) or III (11%–50%), and the vesicle abundances were of grades A₂ or A₃ among different sites. AM fungi produced a large number of mycelia and vesicles in the roots of I. lactea after colonization. Thirty-seven species belonging to 15 genera of AM fungi were isolated from the rhizosphere of I. lactea and identified by morphological identification. Funneliformis and Glomus were the dominant genera, accounting for 21.79% and 20.85% of the total number, respectively. F. mosseae and Rhizophagus intraradices were isolated in all samples with importance values of 58.62 and 51.19, respectively. These results are expected to provide a theoretical basis for the analysis of the salt tolerance mechanism of I. lactea and for the discovery, exploration and further screening of AM fungal resources with salinity tolerances in saline-alkaline soils.     

High abundance and role of antifungal bacteria in compost-treated soils in a wildfire area

Compost has been widely used in order to promote vegetation growth in post-harvested and burned soils. The effects on soil microorganisms were scarcely known, so we performed the microbial analyses in a wildfire area of the Taebaek Mountains, Korea, during field surveys from May to September 2007. Using culture-dependent and -independent methods, we found that compost used in burned soils influenced a greater impact on soil fungi than bacteria. Compost-treated soils contained higher levels of antifungal strains in the genera Bacillus and Burkholderia than non-treated soils. When the antifungal activity of Burkholderia sp. strain O1a_RA002, which had been isolated from a compost-treated soil, was tested for the growth inhibition of bacteria and fungi isolated from burned soils, the membrane-filtered culture supernatant inhibited 19/37 fungal strains including soil fungi, Eupenicillium spp. and Devriesia americana; plant pathogens, Polyschema larviformis and Massaria platani; an animal pathogen, Mortierella verticillata; and an unidentified Ascomycota. However, this organism only inhibited 11/151 bacterial strains tested. These patterns were compatible with the culture-independent DGGE results, suggesting that the compost used in burned soils had a greater impact on soil fungi than bacteria through the promotion of the growth of antifungal bacteria. Our findings indicate that compost used in burned soils is effective in restoring soil conditions to a state closer to those of nearby unburned forest soils at the early stage of secondary succession.     

Desert soil fungi isolated from Saudi Arabia: cultivable fungal community and biochemical production

Desert soils harbor fungi that have survived under highly stressed conditions of high temperature and little available moisture. This study was designed to survey the communities of cultivable fungi in the desert soils of the Arabian Peninsula and to screen the fungi for the potentially valuable antioxidants (flavonoids, phenols, saponins, steroids, tannins, terpenoids, and alkaloids) and enzymes (cellulase, laccase, lipase, protease, amylase, and chitinase). Desert soil was sampled at 30 localities representing different areas of Saudi Arabia and studied for physico-chemical soil properties. Five types of soil texture (sand, loamy sand, sandy loam, silty loam, and sandy clay loam) were observed. A total of 25 saprotrophic species was identified molecularly from 68 isolates. Our survey revealed 13 culturable fungal species that have not been reported previously from Arabian desert soils and six more species not reported from Saudi Arabian desert soils. The most commonly recorded genera were Aspergillus (isolated from 20 localities) and Penicillium (6 localities). The measurements of biochemicals revealed that antioxidants were produced by 49 and enzymes by 52 isolates; only six isolates did not produce any biochemicals. The highest biochemical activity was observed for the isolates Fusarium brachygibbosum and A. phoenicis. Other active isolates were A. proliferans and P. chrysogenum. The same species, for instance, A. niger had isolates of both high and low biochemical activities. Principal component analysis gave a tentative indication of a relationship between the biochemical activity of fungi isolated from soil and soil texture variables namely the content of silt, clay and sand. However, any generalizable relation between soil properties and fungal biochemical activities cannot be suggested. Each fungal isolate is probable to produce several antioxidants and enzymes, as shown by the correlation within the compound groups. Desert soil warrants further research as a promising source of biochemicals.     

Arbuscular mycorrhizal status of spring ephemerals in the desert ecosystem of Junggar Basin, China

A survey was made of the arbuscular mycorrhizal (AM) status of 73 spring ephemeral plant species that grow in the desert ecosystem of Junggar Basin, northwest China. The proportion of AM colonization ranged from 7 to 73% with a mean value of 30%. A total of 65 plant species studied were AM with coils/arbuscules or vesicles and the remaining eight species were possibly AM with no coils/arbuscules or vesicles but with fungal mycelia in the root cortex. AM fungal spores were isolated from rhizosphere samples of all 73 plant species and identified. The mean spore density was 22 per 20 ml of air-dried soil, ranging from 0 to 120. Colonization and spore density of perennials were slightly higher than of annuals and varied among different plant families. A total of 603 AM fungal spore (or sporocarp) specimens were isolated belonging to six genera, Acaulospora, Archaeopora, Entrophospora, Glomus, Paraglomus, and Scutellospora; Glomus was the dominant genus. We conclude that spring ephemerals may be highly dependent on AM associations for survival in the very infertile and arid soils of this desert ecosystem.Electronic supplementary material Supplementary material is available for this article at