Effects of soil temperature and anaerobiosis on degradation of biodegradable plastics in soil and their degrading microorganisms

The bioplastic PHB/HV (copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate) underwent a faster degradation at 30°C than at 52°C in soil under aerobic conditions, while there was no remarkable difference between 30°C and 52°C in the degradation rate of PCL [poly(ε-caprolactone)], PBSA (polybutylene succinate and agipate), and PBS (polybutylene succinate). PHB showed the fastest degree of degradation among the four plastics at 30°C and PBSA the fastest at 52°C. Degradation of all the four plastics was nor observed both at 30°C and 52°C under anaerobic conditions for 50 d. Microorganisms on the degrading plastics appeared to be diverse at 30"C, including bacteria and fungi. However, among the several to ca. 10 kinds of bacterial and fungal strains isolated from the degradation sites of each plastic at 30°C, only one or two fungal strains were able to degrade the respective plastics in vitro. The degraders were identified as Mucor sp. (PHB), Paecilomyces sp. (PCL), Aspergillus sp. (PBSA), and Cunninghamella sp. (PBSA). In contrast, only a single type of fungus was observed at the degradation sites of PCL and PBSA at 52°C. The fungus isolated from PCL and PBSA was identified as Thermomyces sp. This study demonstrated that soil temperature and anaerobiosis exerted significant effects on the degradation of the plastics, and that fungi were mainly responsible for the degradation of the plastics in soil.     

Communities of microscopic fungi in contaminated and reference Al-Fe-humus podzols and their influence on copper mobility

The transformation of microscopic fungi communities under the impact of contamination, the influence of environmental conditions (soil temperature and moisture) on the development of fungal communities in the course of model successions and on the copper mobility in soil, and the most favorable conditions for copper mobilization were studied in Al-Fe-humus podzols of the Kola peninsula contaminated with heavy metals (Cu and Ni). The long-term aerotechnogenic contamination affects the structure of the microbial communities; the species diversity of the communities can somewhat increase at the expense of the increasing diversity of rare, atypical, and “weed” species and decreasing numbers of dominants. The dynamics of fungal communities are less expressed in the contaminated soils as compared with the reference soils. The conditions of rather low temperature (5°C) and an increased moisture content (120% of the field water capacity) are the most favorable for mycelium growth and copper mobilization. Such conditions are typical of the soils in the studied region. Hence, there is a possibility for the soil self-purification due to gradual mobilization of the copper and its subsequent leaching.     

A preliminary study of the growth of fungi and bacteria from temperate and antarctic soils in relation to temperature

The growth of fungi isolated from a lowland temperate site (Roudsea Wood National Nature Reserve), an upland temperate moorland (Moor House National Nature Reserve) and an oceanic Antarctic island (Signy, S. Orkneys) was compared at 1, 14 and 25°C. This showed that low temperatures caused greatest retardation of growth in fungi from the warmest site (Roudsea) and least from the coldest site (Signy Island). At Moor House, fungi which were isolated most frequently in winter were able to grow better at 1°C than summer forms. The fungal flora of Signy Island was restricted and consists of cold tolerant cosmopolitan species which have been selected by or become adapted to the prevailing low temperatures. Of fungi isolated from any two of the sites, Mortierella alpina and Mucor hiemalis showed temperature adaptation correlated with prevailing site temperature, while Trichoderma viride, Penicillium thomii, and P. frequentans showed no adaptation.     

Comparative Analysis of the Number and Structure of the Complexes of Microscopic Fungi in Tundra and Taiga Soils in the North of the Kola Peninsula

The number, biomass, length of fungal mycelium, and species diversity of microscopic fungi have been studied in soils of the tundra and taiga zones in the northern part of the Kola Peninsula: Al-Fe-humus podzols (Albic Podzols), podburs (Entic Podzols), dry peaty soils (Folic Histosols), low-moor peat soils (Sapric Histosols), and soils of frost bare spots (Cryosols). The number of cultivated microscopic fungi in tundra soils varied from 8 to 328 thousand CFU/g, their biomass averaged 1.81 ± 0.19 mg/g, and the length of fungal mycelium averaged 245 ± 25 m/g. The number of micromycetes in taiga soils varied from 80 to 350 thousand CFU/g, the number of fungal propagules in some years reached 600 thousand CFU/g; the fungal biomass varied from 0.23 to 6.2 mg/g, and the length of fungal mycelium varied from 32 to 3900 m/g. Overall, 36 species of fungi belonging to 16 genera, 13 families, and 8 orders were isolated from tundra soils. The species diversity of microscopic fungi in taiga soils was significantly higher: 87 species belonging to 31 genera, 21 families, and 11 orders. Fungi from the Penicillium genus predominated in both natural zones and constituted 38–50% of the total number of isolated species. The soils of tundra and taiga zones were characterized by their own complexes of micromycetes; the similarity of their species composition was about 40%. In soils of the tundra zone, Mortierella longicollis, Penicillium melinii, P. raistrickii, and P. simplicissimum predominated; dominant fungal species in soils of the taiga zone were represented by M. longicollis, P. decumbens, P. implicatum, and Umbelopsis isabellina.     

Culture,food selection and growth rate in the mycophagous ciliate Grossglockneria acuta Foissner, 1980: First evidence of autochthonous soil ciliates

A culture method with defined medium for the soil ciliate Grossglockneria acuta Foissner, 1980 is described. Food selection studies reveal that it fed exclusively on three out of 11 fungi tested (Mucor mucedo, Mucoraceae and Aspergillus sp.) although all have a chitinous cell wall. The rejected species may synthesize antiprotozoal substances. No growth was obtained with yeast, bacteria, flagellates or the ciliate Colpoda aspera Kahl. Under laboratory conditions generation times ranged from 34.25 h (4.5°C) to 3.86 h (30°C). Correspondingly, population growth rate values ranged between 0.036 h^?1 up to 0.323 h^?1. A significant increase in population growth rate was observed between 5.5° and 21°C, whereas there was no reproduction at 40°C. The most pronounced acceleration in population growth rate occurred between 10° and 21°C. This is near the highest mean monthly temperature of the natural habitat (16.7°C) of our population. Field observations yielded a higher density and frequency of G. acuta in alpine soils than in lowland ones. The experiments suggest that the low annual mean temperature could be responsible for this because highest individual densities develop at 4.5°C. The highly specialized diet and the oral structure—a tentacle which is used in breaking up and sucking out hyphae and spores—are convincing proof that G. acuta is autochthonous to soil. This is emphasized by the fact that we could not find any member of the family Grossglockneridae during the investigation of more than 200 running and stagnant waters.     

Fungal colonization of phyllosphere and litter of<Emphasis Type="Italic"> Quercus rotundifolia</Emphasis> Lam. in a holm oak forest (High Atlas,Morocco)

The microfungal flora of holm oak living, senesced and litter leaves was studied at five different stages of decomposition using three different isolation methods. Holm oak leaves are first colonized on the tree by a variety of primary saprophytes such as Trichothecium, Aureobasidium, Cladosporium, Epicoccum and Alternaria. After leaf fall there is an intensive development of the fungal flora, including both species already present in the phyllosphere and new colonizers from the litter layer. With increasing decomposition initial colonizers gradually disappear, being replaced by other forms. When all isolation methods were pooled, maximum biodiversity (species richness) of the fungus flora was observed during the first three stages of leaf litter decomposition, but strong variation occurred according to the isolation method. Sterilization of the leaf material revealed that a number of fungal strains were present inside the holm oak leaves before abscission, increasing from living to senescent stages, and that a strong decrease in the internal colonization of leaf litter was observed at late decomposition stages.     

Soil temperature,mycorrhizal infection and nodulation of Medicago truncatula and Trifolium subterraneum

Establishment of vesicular-arbuscular mycorrhizal fungi in plant roots involves a pre-infection phase of propagule germination, hyphal growth and appressorium formation, followed by growth of the fungus within the root. The effect of soil temperature on the pre-infection stage was examined by counting the numbers of fungal “entry-points” on the main roots of Medicago truncatula and Trifolium subterraneum, grown at soil temperatures of 12°, 16°, 20° and 25°C for periods up to 12 days. Increased root temperature was positively associated with increased numbers of “entry-points”. This effect was more marked between 12° and 16°C than at higher temperatures, as shown by comparing plants at the same stage of development (emergence of spade leaf) and by calculating the results as entry points per cm root.The first root nodules appeared sooner at higher temperatures (20° and 25°), but subsequent development of nodules (measured as nodule number and aggregate volume of nodules per plant, up to 21 days) was best at 16°C for both host Rhizobium combinations in non-sterile and autoclaved soil. There was no evidence that competition between mycorrhizal fungi and Rhizobium for infection sites occurred.A method of obtaining numbers of infective propagules of vesicular-arbuscular mycorrhizal fungi in soil is described.     

Global diversity and distribution of arbuscular mycorrhizal fungi

Arbuscular mycorrhizal (AM) fungi form associations with most land plants and can control carbon, nitrogen, and phosphorus cycling between above- and belowground components of ecosystems. Current estimates of AM fungal distributions are mainly inferred from the individual distributions of plant biomes, and climatic factors. However, dispersal limitation, local environmental conditions,and interactions among AM fungal taxa may also determine local diversity and global distributions. We assessed the relative importance of these potential controls by collecting 14,961 DNA sequences from 111 published studies and testing for relationships between AM fungal community composition and geography, environment, and plant biomes. Our results indicated that the global species richness of AM fungi was up to six times higher than previously estimated, largely owing to high beta diversity among sampling sites. Geographic distance, soil temperature and moisture, and plant community type were each significantly related to AM fungal community structure, but explained only a small amount of the observed variance. AM fungal species also tended to be phylogenetically clustered within sites, further suggesting that habitat filtering or dispersal limitation is a driver of AM fungal community assembly. Therefore, predicted shifts in climate and plant species distributions under global change may alter AM fungal communities.     

Mycological complexes in Holocene and Late Pleistocene paleohorizons and in fragments of paleosols

Fragments of buried Late Pleistocene (30000-year-old) and Early Holocene (10000-year-old) paleosols contained viable complexes of microscopic fungi. The mycobiota of these paleosols represents a pool of fungal spores that is lower in number and species diversity as compared to that in the recent humus horizons and higher than that in the inclosing layers. The central part of the paleosol profiles is greatly enriched in microscopic fungi. In the intact humus horizons of the Late Holocene (1000–1200 years) paleosols, actively functioning fungal complexes are present. These horizons are characterized by their higher level of CO₂ emission. The buried horizons, as compared to the recent mineral ones, contain a greater fungal biomass (by several times) and have a higher species diversity of microscopic fungi (including fungi that are not isolated from the recent horizons). Nonsporulating forms are also present there as sterile mycelium. The seasonal dynamics of the species composition and biomass of the fungal complexes were more prominent and differed from those inherent to the surface soil horizons. In the buried humus horizons, the dynamics of the fungal biomass were mainly due to the changes in the content of spores. The data on the composition of the fungal complexes in the buried soils confirm (due to the presence of stenotopic species) the results of paleobotanic analyses of the past phytocenoses or do not contradict them.     

Effect of temperature and soil moisture on the survival and symbiotic effectiveness of Frankia spp.

Comparison of the effects of temperature on the growth in culture (increase in protein) of Frankia showed that three strains isolated from Casuarina were more tolerant of high temperature (45°C) than a strain from Alnus rubra. Optimal temperatures for growth of the Casuarina strains were in the range 25–30°C. Growth of the Alnus strain was good at 25°C but poor at 37°C. High temperatures (35–40°C) during storage for 7 months of these Frankia strains in sand, inoculated initially with liquid culture or with Frankia incorporated into alginate beads and permitted to dry, resulted in substantial loss of infectivity for the host plant species. Loss in infectivity was greater with an Alnus Frankia strain than strains from Casuarina cunninghamiana, C. equisetifolia and C. junghuniana. Three Frankia strains from C. equisetifolia were incorporated into a sand/perlite mixture with three different moisture regimes (field moisture capacity – wet: watered and maintained at field capacity; watered to field capacity but then allowed to dry – moderately wet; or watered to half field capacity and then permitted to dry – dry) and then stored for 12 weeks at 25°C and 35°C. Assessment by the most probable number (MPN) technique of the infectivity of the sand mixture for nodulation of C. equisetifolia showed significant interactions between Frankia strain, temperature and soil moisture content. The infectivity of Frankia strains ORS020607 and UGL020602q was not affected by incubation in wet sand at 25°C but fell by more than half after 12 weeks in moderate and dry conditions. Changes in infectivity were similar when incubation was at 35°C. By contrast, the infectivity of UGL020603q fell substantially under all moisture conditions and at both temperatures. The data show the importance of screening for tolerance of both temperature and moisture content when selecting strains for preparation of inoculum for use in hot climates. Received: 25 January 1996     

A computer simulation model for soil temperatures in Australian cereal cropping

The effect of tillage on soil temperature and the effects of temperature on plant performance have been investigated by various authors, but soil temperature data are not available in many of Australia's cropping areas. A model is therefore needed to simulate suitable temperature data for research and management applications. A suitable computer model using readily available soil and climatic data to calculate hourly soil temperatures is presented for this purpose. The model was identified in the literature and modified for use with a Ug5.2 clay soil under conditions of bare fallow and with a wheat crop. Field data for modifying and validating the model were obtained from a trial site in the Wimmera wheat growing region of Victoria, Australia. Validation results for the modified model (designated the ASTC model) are presented, based on recorded temperatures over a 1-month period on the surface and at 50 mm depth in the soil profile. The model predicted the soil temperature at 50 mm depth under a bare surface with a standard error of estimate (SEE) of 2.4°C during validation. The corresponding value with a wheat crop was 2.1°C. While simulating the entire period of recorded temperatures the ASTC model had average hourly prediction errors of 2.1°C with the bare fallow (4-month period) and 1.1°C with the wheat crop (4-month period) at 50 mm depth. The standard errors of estimate for the predicted temperatures in the extended application of the model varied from 1.9°C at 50 mm depth to 0.2°C at 500 mm depth with the wheat crop. Corresponding SEE values with the bare fallow ranged from 3.0 to 0.4°C.     

Abundance and composition response of wheat field soil bacterial and fungal communities to elevated CO<Subscript>2</Subscript> and increased air temperature

The abundance and diversity of soil bacterial and fungal communities in a wheat field under elevated atmospheric CO₂ concentrations and increased air temperatures were investigated using qPCR and pyrosequencing. Elevated CO₂ concentrations significantly increased the abundances of bacteria and fungi, and an increase of air temperatures significantly reduced fungal abundance. We found that Proteobacteria, Bacteroidetes, Chloroflexi, and Ascomycota were the most abundant bacterial and fungal phyla in the wheat field soil. Elevated CO₂ concentrations and increased air temperatures had no significant effect on the bacterial alpha diversity, whereas fungal richness was reduced under warming treatments. Moreover, we note that certain bacterial and fungal groups responded differentially to elevated CO₂ concentrations and increased air temperatures, and fungal species were highly sensitive to climatic changes.     

微生物响应PBAT-PLA生物降解膜袋工业需氧堆肥降解机制

塑料污染已对全球环境造成严重威胁,生物降解塑料的推广使用及其工业堆肥是治理塑料污染的有效途径之一。该研究根据标准GB/T 19277.1-2011,在（58±2）℃的特殊高温条件下,对PBAT-PLA生物降解膜袋进行有氧堆肥降解,并选择微晶纤维素作为对照。通过对堆肥中的微生物进行16S/18S高通量测序,分析降解过程中细菌/真菌的群落种类和数量变化,包括物种多样性、物种组成、物种差异分析、样本比较分析,并结合扫描电镜下的微观形貌,深入探寻可降解塑料膜袋在工业需氧堆肥过程中的微生物响应降解机制。结果表明：微晶纤维素和生物降解膜袋在降解活跃期（第140天取样）,其所在堆肥中大量存在的优势菌属为Sphaerobacter（球杆菌属,放线菌纲）,分别占比20.25%和39.44%。与同样条件下不含降解材料的对照组堆肥相比,微晶纤维素/生物降解膜袋工业需氧堆肥降解过程中显著增长的4种菌属中有3种属于放线菌,说明放线菌对聚酯物的解聚以及纤维素的降解具有积极的作用。试验结果也表明了聚酯和纤维素的完整生物降解过程不依赖单一菌种,而是微生物协同作用的结果。     

Soil structure and soil-borne diseases: using epidemiological concepts to scale from fungal spread to plant epidemics

Many epidemics of root diseases involving soil fungi depend on the interplay between fungal growth and the spatial and temporal heterogeneity of the soil environment. Colonization or infection of a root occurs at fine scales with growth and movement of fungal mycelia through soil. However, epidemics are observed at coarser scales, and depend on a cascading spread through populations of roots. We briefly review conventional analyses of soil‐borne epidemics and argue that these treat soil physical conditions at scales too coarse to be meaningful for interactions between soil, plants and fungi, and fail to consider the effect of soil physical conditions on the underlying epidemiological processes. Instead, we propose a conceptual epidemiological framework that integrates spatial scales and use this to review the effect of soil structure on the dynamics of soil‐borne pathogenic fungi. Using the soil‐borne fungal plant pathogen Rhizoctonia solani as an example, we demonstrate that invasion of fungi into host populations is critically affected by environmental conditions operating at each of two scales: (i) at the microscopic scale (μm ? cm) the fungus preferentially explores certain pathways in soil, and small changes in soil physical conditions make the fungus switch from small, dense colonies to large, sparse and rapidly expanding ones; (ii) at the larger scale (cm ? dm) a critical density of susceptible hosts is required, in excess of which fungi switch from non‐invasive to invasive spread. Finally, we suggest that the approach will increase the applicability of research dealing with microscopic soil–plant–microbe interactions towards the solution of large‐scale epidemiological problems.     

Temperature sensitivity of gross N transformation rates in an alpine meadow on the Qinghai-Tibetan Plateau

Purpose

Alpine ecosystems on the Qinghai-Tibetan Plateau are sensitive to global climatic changes. However, the effects of temperature change resulting from global warming or seasonal variation on soil N availability in those ecosystems are largely unknown.

Materials and methods

We therefore conducted a ¹⁵N tracing study to investigate the effects of various temperatures (5–35 °C) on soil gross N transformation rates in an alpine meadow (AM) soil on the Qinghai-Tibetan Plateau. A natural secondary coniferous forest (CF) soil from the subtropical region was chosen as a reference to compare the temperature sensitivity of soil gross N transformation rates between alpine meadow and coniferous forest.

Results and discussion

Our results showed that increasing temperature increased gross N mineralization and NH₄ ⁺ immobilization rates and overall enhanced N availability for plants in both soils. However, both rates in the CF soil were less sensitive to a temperature change from 5 to 15 °C compared to the AM soil. In both soils, different N retention mechanisms could have been operating with respect to changing temperatures in the different climatic regions. In the CF soil, the absence of NO₃ ^? production at all incubation temperatures suggests that in the subtropical soil which is characterized by high rainfall, an increase in N availability due to increasing temperature could be completely retained in soils. In contrast, the AM soil may be vulnerable to N losses with respect to temperature changes, in particular at 35 °C, in which higher nitrification rates were coupled with lower NH₄ ⁺ and NO₃ ^? immobilization rates.

Conclusions

Our results suggest that increased soil temperature arising from global warming and seasonal variations will most likely enhance soil N availability for plants and probably increase the risk of N losses in the alpine meadow on the Qinghai-Tibetan Plateau.

     

Effect of fungicide thiram on microbial communities of rice seed surface estimated by culture-dependent and culture-independent (PCR-RFLP) methods

Coating of rice seeds with fungicide Thiram improved the seed germination capability over a long period of time (11 weeks) under low temperature conditions (4 and 8°C), which simulated the sowing of rice seeds in the winter season (the farmer's slack season). To analyze the effect of Thiram on the community structure of microorganisms on the rice seed surface, culture-dependent and culture-independent (PCR-RFLP) methods were applied. PCR-RFLP patterns of 16S rDNA showed that the bacterial communities on the rice seed surface were different between coated and uncoated treatments under 8°C conditions, but that they were very similar under 4°C conditions. PCR-RFLP patterns of 18S rDNA revealed the remarkable effect of Thiram on the fungal community structure under both 4 and 8°C conditions. Although the fungal communities were quite different between coated and uncoated seeds at the beginning of incubation, the fungal communities on the coated seed surface became similar to those of uncoated seeds along with the duration of the incubation period. As the dominance percentage of Fusarium spp. among the isolates increased with the duration of the incubation period for both coated and uncoated seeds, Fusarium was considered to be a responsible for the poor germination of rice seeds that were sown in the winter season.     

Non nutritional factors affecting the growth of Trichoderma in culture

Species of Trichoderma typical of cool geographic regions possessed lower temperature optima and maxima than species from warm climatic regions. T. viride Pers. ex S.F. Gray and T. polysporum (Link ex Pers.) Rifai had temperature maxima ranging from 28–31°C and grew much better than other species at 7°C. The temperature maxima of T. koningii Oud. ranged from 32–35°C, T. hamatum (Bon.) Bain. from 30–35°C, T. harzianum Rifai from 30–38°C and T. pseudokoningii Rifai and T. saturnisporum Hammill from 40–41°C.Isolates of Trichoderma varied in their responses to CO₂-enriched atmospheres and the response was pH dependent. The largest responses were obtained on a medium of pH 7.5 as compared to an acid medium of pH 4.4. As the CO₂ level increased from 2 to 10 per cent, growth was reduced on an acid medium and increased on the alkaline medium as compared to cultures incubated in air. In air the optimum pH for Trichoderma ranged between 3–7 and 4.7. However, as the CO₂ concentration was increased to 10 per cent, growth was nearly as good at pH 7.5 as at pH 4.4.The addition of HCO₃^? to an alkaline medium strongly inhibited Trichoderma while having little effect on fungi isolated from roots or a strongly alkaline habitat. Low moisture content of litter was shown to have a variable effect on Trichoderma species and may be important in the ecology of Trichoderma. Salt (NaCl) concentration is not considered to be an important ecological factor.     

Mineralization of bacteria and fungi in chloroform-fumigated soils

It has been suggested by others that the size of the flush of mineralization caused by CHC1₃ fumigation can be used to estimate the amount of microbial biomass in soils. Calculation of biomass from the flush requires that the proportion of CHCl₃-killed cell C mineralized be known. To determine this proportion, 15 species of [¹⁴C]labelled fungi and 12 species of [¹⁴C]labelled bacteria were added to four types of soil and these were fumigated for 24 h with CHC1₃, reinoculated with unfumigated soil, and incubated at 22°C for 10 days. The average percentage mineralization of the fungi was 43.7 ± 5.3, while the average for the bacteria was 33.3 ± 9.9. Using a 1:3 ratio for distribution of total biomass between the bacterial and fungal populations, respectively, it was calculated that the average mineralization of both types of cells was 41.1%. In experiments conducted to determine if CHC1₃ vapour alters stabilized microbial metabolites or dead microbial cells in a manner which makes them more susceptible to degradation, it was found that both fumigated and unfumigated dead fungal materials mineralized to the same extent in soil during 10 days of incubation.     

The mycoflora of Eucalyptus maculata leaf litter

The mycoflora of the litter of Eucalyptus maculata was studied using three isolation methods. A total of 45 species, representing 22 genera were isolated from the various litter horizons. A comparison between the fungal flora of the litter (L) and that of the underlying mineral soil (A₁) was also made. It was found that 39 per cent of the species were common to both the forest litter and the soil proper. The composition of the litter fungal flora, however, differed from that of the underlying mineralized soil.     

Stabilisation of soil against wind erosion by six saprotrophic fungi

Soils with biological crusts that consist of fungal hyphae, bacteria and other small organisms usually resist erosion. However, the processes by which soil organisms stabilise air-dry aggregates against wind erosion are not well understood. We used saprotrophic fungi to examine some of these processes in a sandy clay loam (Hypercalcic Calcarosol). Soil aggregates, wetted with distilled water or glucose solution, were inoculated with one of six fungi, and incubated in darkness at 24 °C for 7 d in petri dishes under sterile conditions. Abrasion resistance (taken as resistance to wind erosion), tensile strength of soil, hot-water extractable carbohydrates (HWEC), dispersion index, pH, and hyphal length density (HLD) were each measured across all treatments. In all treatments, stability (abrasion resistance) and tensile strength, were positively related to HLD. Such relationships have not been reported elsewhere. All fungi enlarged the aggregates of the soil by cross-linkage and entanglement of particles, but with different processes, or different intensity of the processes, between species (for the same amount of substrate). The skins seen in scanning electron micrographs of stabilised soil were probably extracellular polysaccharides also produced by the fungi. We propose that the ductile failure of disks of soil, particularly those inoculated with Mucor sp., under tensile stress was due to movement of enmeshed particles, whereas the brittle failure of disks of soil inoculated with the other fungal species was due to metabolites or dispersed clay on the surface of the hyphae which limited deformation.