Novel Symbiotic Protoplasts Formed by Endophytic Fungi Explain Their Hidden Existence,Lifestyle Switching,and Diversity within the Plant Kingdom

Diverse fungi live all or part of their life cycle inside plants as asymptomatic endophytes. While endophytic fungi are increasingly recognized as significant components of plant fitness, it is unclear how they interact with plant cells; why they occur throughout the fungal kingdom; and why they are associated with most fungal lifestyles. Here we evaluate the diversity of endophytic fungi that are able to form novel protoplasts called mycosomes. We found that mycosomes cultured from plants and phylogenetically diverse endophytic fungi have common morphological characteristics, express similar developmental patterns, and can revert back to the free-living walled state. Observed with electron microscopy, mycosome ontogeny within Aureobasidium pullulans may involve two organelles: double membrane-bounded promycosome organelles (PMOs) that form mycosomes, and multivesicular bodies that may form plastid-infecting vesicles. Cultured mycosomes also contain a double membrane-bounded organelle, which may be homologous to the A. pullulans PMO. The mycosome PMO is often expressed as a vacuole-like organelle, which alternatively may contain a lipoid body or a starch grain. Mycosome reversion to walled cells occurs within the PMO, and by budding from lipid or starch-containing mycosomes. Mycosomes discovered in chicken egg yolk provided a plant-independent source for analysis: they formed typical protoplast stages, contained fungal ITS sequences and reverted to walled cells, suggesting mycosome symbiosis with animals as well as plants. Our results suggest that diverse endophytic fungi express a novel protoplast phase that can explain their hidden existence, lifestyle switching, and diversity within the plant kingdom. Importantly, our findings outline “what, where, when and how”, opening the way for cell and organelle-specific tests using in situ DNA hybridization and fluorescent labels. We discuss developmental, ecological and evolutionary contexts that provide a robust framework for continued tests of the mycosome phase hypothesis.     

Volatile Emissions from an Epiphytic Fungus are Semiochemicals for Eusocial Wasps

Microbes are ubiquitous on plant surfaces. However, interactions between epiphytic microbes and arthropods are rarely considered as a factor that affects arthropod behaviors. Here, volatile emissions from an epiphytic fungus were investigated as semiochemical attractants for two eusocial wasps. The fungus Aureobasidium pullulans was isolated from apples, and the volatile compounds emitted by fungal colonies were quantified. The attractiveness of fungal colonies and fungal volatiles to social wasps (Vespula spp.) were experimentally tested in the field. Three important findings emerged: (1) traps baited with A. pullulans caught 2750?% more wasps on average than unbaited control traps; (2) the major headspace volatiles emitted by A. pullulans were 2-methyl-1-butanol, 3-methyl-1-butanol, and 2-phenylethyl alcohol; and (3) a synthetic blend of fungal volatiles attracted 4,933?% more wasps on average than unbaited controls. Wasps were most attracted to 2-methyl-1-butanol. The primary wasp species attracted to fungal volatiles were the western yellowjacket (Vespula pensylvanica) and the German yellowjacket (V. germanica), and both species externally vectored A. pullulans. This is the first study to link microbial volatile emissions with eusocial wasp behaviors, and these experiments indicate that volatile compounds emitted by an epiphytic fungus can be responsible for wasp attraction. This work implicates epiphytic microbes as important components in the community ecology of some eusocial hymenopterans, and fungal emissions may signal suitable nutrient sources to foraging wasps. Our experiments are suggestive of a potential symbiosis, but additional studies are needed to determine if eusocial wasp–fungal associations are widespread, and whether these associations are incidental, facultative, or obligate.     

Psychrophilic and Mesophilic Fungi in Fruit-Filled Pastries

Surveys of the mold flora of frozen blueberry and cherry pastries were undertaken. Molds were enumerated by preparing pour plates of the blended product and incubating the plates at 0, 5, 10, and 20 C. In this manner, the total fungal content of the product could be ascertained from the 10 and 20 C plates, and the psychrophilic fungal population was represented by those fungi which grew at 0 and 5 C. The pastry portion, or crust, of the blueberry material was sampled separately from the filling portion. Certain differences in fungal flora were apparent. Aureobasidium pullulans was the dominant fungus in crust at all temperatures of isolation. However, Penicillium thomii proved to be the most common mesophilic fungus in the filling portion, and A. pullulans was the most common psychrophile in the filling. Aspergilli were quite common in the crust, but, in general, were absent from the fruit filling. Cherry pastries had a much smaller total fungal flora than did the blueberry product. However, A. pullulans again was the most prevalent fungus in cherry pastries at all temperatures of isolation. Certain differences in fungal flora were apparent in the two fruit products. Phoma spp. were almost completely absent in blueberries, but represented the second most common fungus in cherry pastries. Blueberry filling had 440 psychrophilic fungi per gram of sample (at 0 C), blueberry crust had 65 per gram, and cherry pastries had 77 per gram.     

Biological control of strawberry crown rot,root rot and grey mould by the beneficial fungus Aureobasidium pullulans

Utilization of biocontrol agents is a sustainable approach to reduce plant diseases caused by fungal pathogens. In the present study, we tested the effect of the candidate biocontrol fungus Aureobasidium pullulans (De Bary) G. Armaud on strawberry under in vitro and in vivo conditions to control crown rot, root rot and grey mould caused by Phytophthora cactorum (Lebert and Cohn) and Botrytis cinerea Pers, respectively. A dual plate confrontation assay showed that mycelial growth of P. cactorum and B. cinerea was reduced by 33–48% when challenged by A. pullulans as compared with control treatments. Likewise, detached leaf and fruit assays showed that A. pullulans significantly reduced necrotic lesion size on leaves and disease severity on fruits caused by P. cactorum and B. cinerea. In addition, greenhouse experiments with whole plants revealed enhanced biocontrol efficacy against root rot and grey mould when treated with A. pullulans either in combination with the pathogen or pre-treated with A. pullulans followed by inoculation of the pathogens. Our results demonstrate that A. pullulans is an effective biocontrol agent to control strawberry diseases caused by fungal pathogens and can be an effective alternative to chemical-based fungicides.

     

Exploring the biocontrol potential of fungal endophytes from an Andean Colombian Paramo ecosystem

We studied the diversity and biocontrol potential of 100 fungal endophytes isolated from Espeletia spp., endemic plant species from the Paramo in the Andean mountain range. Our sample was genotypically highly diverse at all ITS similarity levels. The antagonistic properties of these isolates were tested against common crop pathogens in Colombia, including Pectobacterium carotovorum, Ralstonia solanacearum, Pseudomonas syringae, Xanthomonas campestris, Rhizoctonia solani, Botrytis cinerea, Fusarium oxysporum, and Phytophthora infestans. All endophytic isolates were able to significantly inhibit the growth of at least one of the plant pathogens tested (P?<?0.05). Three main types of endophyte/pathogen interactions were observed. However, only those endophytes that produced an evident inhibition halo were further studied using their crude extracts to confirm that the inhibitory effect was due to the production of endophytic bioactive metabolites. From these experiments, nine promising isolates were selected for co-inoculation tests with R. solani in tomato plants. The isolates identified as Aureobasidium pullulans and Paraconiothyrium sporulosum not only protected the plants against this pathogen but also allowed them to exhibit similar growth and development as the uninoculated control. This work explores new alternatives for disease management without the application of chemical pesticides.     

Fungal endophytes from cactus Cereus jamacaru in Brazilian tropical dry forest: a first study

Endophytic fungi live within the healthy tissues of plants and can promote host species tolerance to different environmental stresses. However, most studies have been of plants in humid environments, and there are few reports of the benefits of such associations for plants of extreme environments. Our aims were to identify endophytic fungi using morphological taxonomy, to explore richness and estimate species frequency in a cactus, C. jamacaru, in Brazilian tropical dry forest (Caatinga). We thus identified 59 taxa, corresponding to 69.7 % of the total number of isolates; the other 30.3 % were sterile mycelia. Cladosporium cladosporioides and Fusarium oxysporum were the species most commonly isolated, followed by Acremonium implicatum, Aureobasidium pullulans, Trichoderma viride, Chrysonilia sitophila, and Aspergillus flavus. Forty-seven species are recorded for the first time as endophytic fungi of cacti, and 18 others as endophytes for Brazil; this suggests that C. jamacaru harbors a highly diverse fungal community as measured by a diversity index. However, species accumulation curves suggest that our study still underestimates endophyte diversity because it does not provide an exhaustive sample. To our knowledge, this is the first report of endophytic fungi from C. jamacaru in tropical dry forests.     

Emergence of <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> as human fungal pathogen and molecular assay for future medical diagnosis

Despite the great importance of Aureobasidium pullulans in biotechnology, the fungus had emerged as an opportunistic human pathogen, especially among immunocompromised patients. Clinical detection of this rare human fungal pathogen presently relies on morphology diagnosis which may be misleading. Thus, a sensitive and accurate quantitative molecular assay for A. pullulans remains lacking. In this study, we presented the microscopy observations of A. pullulans that reveals the phenotypic plasticity of the fungus. A. pullulans-specific primers and molecular beacon probes were designed based on the fungal 18S ribosomal RNA (rRNA) gene. Comparison of two probes with varied quencher chemistry, namely BHQ-1 and Tamra, revealed high amplification efficiency of 104% and 108%, respectively. The optimized quantitative real-time PCR (qPCR) assays could detect and quantify up to 1 pg concentration of A. pullulans DNA. Both assays displayed satisfactory performance parameters at fast thermal cycling mode. The molecular assay has great potential as a molecular diagnosis tool for early detection of fungal infection caused by A. pullulans, which merits future study in clinical diagnosis.     

Short-term impacts of soil freeze-thaw cycles on roots and root-associated fungi of Holcus lanatus and Calluna vulgaris

Background and aims

Soil freeze-thaw cycle (FTC) regimes are altered by climate change and known to influence nutrient cycling and plant growth. Here, we explore mechanistic explanations for the changing plant performance of the grass Holcus lanatus and the dwarf shrub Calluna vulgaris.

Methods

144 plant-soil mesocosms were subjected to different FTC-regimes in a climate chamber. Root injury, fungal activity and fungal composition (ITS-sequencing) were quantified.

Results

The applied FTC-scenarios increased root injury by 23% on average while no strong differences between scenarios was found. Root damage was greater in C. vulgaris than in H. lanatus. Fungal activity increased due to the FTC-manipulation and was higher in the C. vulgaris samples, although activity was generally low. No significant shift in the fungal community composition was found immediately after the applied FTCs. A saprobic (Aureobasidium pullulans) and a potentially mycorrhizal fungus (Sebacinales) showed opposing responses to the FTC-manipulation in the different host plants, while a potential phytopathogen (Callophora) decreased in frequency.

Conclusions

Increased fungal activity within these samples is suggested to be related to an increased dominance of saprobic taxa, but not to a shift in qualitative community composition. Single pathogenic species entering the plants through the observed root injuries subsequent to FTC treatments however, may alter plant performance. While these results suggest the importance of root injury for the response of vegetation to FTCs, fungal activity and pathogenic infection need to be further studied under field conditions over longer time periods.     

Biocontrol potential of endophytes harbored in Radula marginata (liverwort) from the New Zealand ecosystem

Radula marginata and Cannabis sativa L. are two phylogenetically unrelated plant species containing structurally similar secondary metabolites like cannabinoids. The major objective of our work was the isolation, identification, biocontrol efficacies, biofilm forming potential and anti-biofilm ability of endophytic microbial community of the liverwort R. marginata, as compared to bacterial endophytic isolates harbored in C. sativa plants. A total of 15 endophytic fungal and 4 endophytic bacterial isolates were identified, including the presence of a bacterial endosymbiont within an endophytic fungal isolate. The endosymbiont was visible only when the fungus containing it was challenged with two phytopathogens Botrytis cinerea and Trichothecium roseum, highlighting a tripartite microbe–microbe interaction and biocontrol potency of endophytes under biotic stress. We also observed sixteen types of endophytic fungal-pathogen and twelve types of endophytic bacterial-pathogen interactions coupled to varying degree of growth inhibitions of either the pathogen or endophyte or both. This showed the magnitude of biocontrol efficacies of endophytes in aiding plant fitness benefits under different media (environmental) conditions. Additionally, it was ecologically noteworthy to find the presence of similar endophytic bacterial genera in both Radula and Cannabis plants, which exhibited similar functional traits like biofilm formation and general anti-biofilm activities. Thus far, our work underlines the biocontrol potency and defensive functional traits (in terms of antagonism and biofilm formation) of endophytes harbored in liverwort R. marginata as compared to the endophytic community of phylogenetically unrelated but phytochemically similar plant C. sativa.     

Interaction of Population Levels of Fusarium oxysporum f. sp. vasinfectum and Meloidogyne incognita on Cotton

In autoclaved greenhouse soil without Fusarium oxysporum f. sp. vasinfectum, Meloidogyne incognita did not cause leaf or vascular discoloration of 59-day-old cotton plants. Plants had root galls with as few as 50 Meloidogyne larvae per plant. Root galling was directly proportional to the initial nematode population level. Fusarium wilt symptoms occurred without nematodes with 77,000 fungus propagules or more per gram of soil. As few as 50 Meloidogyne larvae accompanying 650 fungus propagules caused Fusarium wilt. With few exceptions, leaf symptoms appeared sooner as numbers of either or both organisms increased. In soils infested with both organisms, the extent of fungal invasion and colonization was well correlated with the extent of nematode galling and other indications of the Fusarium wilt syndrome.     

Interaction of abscisic acid and indole-3-acetic acid-producing fungi with Salix leaves

The molds Botrytis cinerea, Cladosporium cladosporioides, and the yeast Aureobasidium pullulans, isolated from the leaves of three short-rotation Salix clones, were found to produce indole-3-acetic acid (IAA). Abscisic acid (ABA) production was detected in B. cinerea. The contents of IAA and ABA in the leaves of the Salix clones and the amounts of fungal propagules in these leaves were also measured, in order to evaluate whether the amounts of plant growth regulators produced by the fungi would make a significant contribution to the hormonal quantities of the leaves. The content of ABA, and to a lesser degree that of IAA, showed a positive correlation with the frequency of infection by the hormone-producing organisms. The amounts of hormone-producing fungi on leaves that bore visible colonies were, however, not sufficiently high to support the claim that either the fungal production of ABA or IAA would significantly contribute to the hormonal contents of the leaves of the Salix clones. It is therefore suggested that the effect of fungal IAA production on plants is limited to the rhizosphere and that B. cinerea, which is a known pathogen, induces ABA production by the mother plant as a response to physiological stress.Abbreviations ABA abscisic acid - ABA-Me abscisic acid methyl ester - GC-MS-SIM gas chromatography-selected ion monitoring-mass spectrometry - IAA indole-3-acetic acid - IAA-Me indole-3-acetic acid methyl ester Author for correspondence.     

Fungus propagules in plastids: the mycosome hypothesis

A stress-induced "mycosome" phase of Aureobasidium pullulans consisting of minute reproductive propagules that may revert directly to walled yeast cells is described. Mycosomes detected by light- and electron-microscopy reproduce within senescent plant plastids, and display three developmental pathways: wall-less cells (protoplasts), yeast cells, or membrane-bounded spherules that harbor plastids. Widespread in plant and algal cells, mycosomes are produced by both ascomycete and basidiomycete fungi. Electronic Publication     

Extracellular fungal polyol lipids: A new class of potential high value lipids

Extracellular fungal glycolipid biosurfactants have attracted attention because productivities can be high, cheap substrates can be used, the molecules are secreted into the medium and the downstream processing is relatively simple. Three classes of extracellular fungal glycolipid biosurfactants have provided most of the scientific advances in this area, namely sophorolipids, mannosylerythritol lipids and cellobioselipids. Polyol lipids, a fourth class of extracellular fungal glycolipid biosurfactants, comprise two groups of molecules: liamocins produced by the yeast-like fungus Aureobasidium pullulans, and polyol esters of fatty acids, produced by some Rhodotorula yeast species. Both are amphiphilic, surface active molecules with potential for commercial development as surfactants for industrial and household applications. The current knowledge of polyol lipids highlights an emerging group of extracellular fungal glycolipid biosurfactants and provides a perspective of what next steps are needed to harness the benefits and applications of this novel group of molecules.     

Isolation of New Aureobasidium Strains That Produce High-Molecular-Weight Pullulan with Reduced Pigmentation

New isolates of Aureobasidium pullulans were obtained from plant leaf surfaces gathered in San Diego County. The new fungal isolates were identified as A. pullulans on the basis of the appearance of polymorphic colonies formed on agar plates, the electrophoretic profiles of repeated genomic DNA sequences, and the production of pullulan in shake flask cultures. The isolates showed different degrees of pigmentation. One of the natural isolates was nonpigmented under mock production conditions in liquid culture, but was still able to synthesize a reduced amount of pigment on agar plates at late times. A mutagenic treatment with ethidium bromide produced derivatives of normally pigmented natural isolates that exhibited an increased tendency toward yeastlike growth and reduced pigmentation. Additionally, some of the new isolates and mutant derivatives accumulated pullulan of relatively high molecular weight in the culture broths.     

Effects on plant growth and root-knot nematode infection of an endophytic GFP transformant of the nematophagous fungus Pochonia chlamydosporia

Pochonia chlamydosporia (Pc123) is a fungal parasite of nematode eggs which can colonize endophytically barley and tomato roots. In this paper we use culturing as well as quantitative PCR (qPCR) methods and a stable GFP transformant (Pc123gfp) to analyze the endophytic behavior of the fungus in tomato roots. We found no differences between virulence/root colonization of Pc123 and Pc123gfp on root-knot nematode Meloidogyne javanica eggs and tomato seedlings respectively. Confocal microscopy of Pc123gfp infecting M. javanica eggs revealed details of the process such as penetration hyphae in the egg shell or appressoria and associated post infection hyphae previously unseen. Pc123gfp colonization of tomato roots was low close to the root cap, but increased with the distance to form a patchy hyphal network. Pc123gfp colonized epidermal and cortex tomato root cells and induced plant defenses (papillae). qPCR unlike culturing revealed reduction in fungus root colonization (total and endophytic) with plant development. Pc123gfp was found by qPCR less rhizosphere competent than Pc123. Endophytic colonization by Pc123gfp promoted growth of both roots and shoots of tomato plants vs. uninoculated (control) plants. Tomato roots endophytically colonized by Pc123gfp and inoculated with M. javanica juveniles developed galls and egg masses which were colonized by the fungus. Our results suggest that endophytic colonization of tomato roots by P. chlamydosporia may be relevant for promoting plant growth and perhaps affect managing of root-knot nematode infestations.     

Dark Septate Root Endophytic Fungus <Emphasis Type="Italic">Nectria haematococca</Emphasis> Improves Tomato Growth Under Water Limiting Conditions

The ascomycetous dark septate endophytic (DSE) fungi characterized by their melanized hyphae can confer abiotic stress tolerance in their associated plants in addition to improving plant growth and health. In this study inoculation of the DSE fungus Nectria haematococca Berk. & Broome significantly improved all the plant growth parameters like the plant height, stem girth, leaf characteristics and plant biomass of drought-stressed tomato. Root characters like the total root length, primary root diameter, 2nd order root number and diameter, root hair number and length were also significantly influenced by the fungal inoculation. Nevertheless, N. haematococca inoculation did not affect root colonization by native arbuscular mycorrhizal (AM) fungi and no significant correlation existed between the AM and DSE fungal variables examined. The proline accumulation in shoots of N. haematococca inoculated plants was significantly higher than uninoculated plants. The present study clearly indicates for the first time the ability of the DSE fungus, N. haematococca in inducing the drought stress tolerance and promoting the growth of the host plant under water stress.     

Endophytic fungi from the root tubers of medicinal plant Stephania dielsiana and their antimicrobial activity

The adverse effects of chemical synthetic fungicides on agricultural fields and the environment are driving a need to search for safer and less environmentally harmful plant protectants to move toward more sustainable development of agriculture. The endophytic fungal community associated with the medicinal plant Stephania dielsiana, and its potential for providing antimicrobial secondary metabolites were investigated. A total of 26 isolates of endophytic fungi were obtained, and 21 isolates were identified and classified into eight different genera, including Briansuttonomyces, Glomerella, Pleosporales, Diaporthe, Phoma, Penicillium, Periconia and Colletotrichum, and the most frequent endophytic species obtained were Diaporthe phaseolorum, Penicillium sp., Periconia igniari and Colletotrichum sp. The ethyl acetate (EtOAc) extract of the endophytic fungus Diaporthe phaseolorum Stdif6 displayed the most significant antifungal activity against all tested phytopathogens, with EC₅₀ values ranging from 0.0138 to 0.3103 mg/mL. While the EtOAc extract of the endophytic fungus Penicillium sp. Stdif9 exhibited greater potential for antibacterial activity, with the minimum inhibitory concentration (MIC) values against seven bacteria ranging from 1.25 to 6 mg/mL. The remarkable antimicrobial activity of fungal endophytes suggests that fungal endophytes harbored inside the root tubers of S. dielsiana hold great promise as biocontrol agents against a broad spectrum of economically significant pathogens.     

House Fly (Musca domestica L.) Attraction to Insect Honeydew

House flies are of major concern as vectors of food-borne pathogens to food crops. House flies are common pests on cattle feedlots and dairies, where they develop in and feed on animal waste. By contacting animal waste, house flies can acquire human pathogenic bacteria such as Escherichia coli and Salmonella spp., in addition to other bacteria, viruses, or parasites that may infect humans and animals. The subsequent dispersal of house flies from animal facilities to nearby agricultural fields containing food crops may lead to pre-harvest food contamination with these pathogens. We hypothesized that odors from honeydew, the sugary excreta produced by sucking insects feeding on crops, or molds and fungi growing on honeydew, may attract house flies, thereby increasing the risk of food crop contamination. House fly attraction to honeydew-contaminated plant material was evaluated using a laboratory bioassay. House flies were attracted to the following plant-pest-honeydew combinations: citrus mealybug on squash fruit, pea aphid on faba bean plants, whitefly on navel orange and grapefruit leaves, and combined citrus mealybug and cottony cushion scale on mandarin orange leaves. House flies were not attracted to field-collected samples of lerp psyllids on eucalyptus plants or aphids on crepe myrtle leaves. Fungi associated with field-collected honeydews were isolated and identified for further study as possible emitters of volatiles attractive to house flies. Two fungal species, Aureobasidium pullulans and Cladosporium cladosporioides, were repeatedly isolated from field-collected honeydew samples. Both fungal species were grown in potato dextrose enrichment broth and house fly attraction to volatiles from these fungal cultures was evaluated. House flies were attracted to odors from A. pullulans cultures but not to those of C. cladosporioides. Identification of specific honeydew odors that are attractive to house flies could be valuable for the development of improved house fly baits for management of this pest species.     

Psychrophilic and Mesophilic Fungi in Frozen Food Products

The mold flora of certain frozen pastries and chicken pies was investigated. Molds were determined qualitatively or quantitatively, or both, by preparing pour plates of the blended product and incubating the plates at various temperatures. The mesophilic fungal flora developed on plates incubated at 10 and 20 C, whereas psychrophilic fungi were obtained on plates incubated at 0 and 5 C. About 2,000 cultures of fungi, representing about 100 different species, were isolated from various products. Four different brands of blueberry, two brands of cherry pastries, two brands of apple, and one brand of raspberry pastries were examined. In addition, two brands of chicken pies were studied. Blueberry pastries had a much higher total fungal population than the other products, although different brands of blueberry pastries varied considerably. Blueberry pastries had from 347 to 1,586 psychrophilic fungi per g. Cherry pastries had about 70 to 110 psychrophiles per g, and apple pastries had 19 to 92 psychrophiles per g. Chicken pies contained very few psychrophilic fungi, about 15 per g. Aureobasidium pullulans was recovered most frequently. About 90% of the psychrophilic fungi found in blueberry products was A. pullulans. Depending upon the brand of cherry pastry, either Phoma spp. or A. pullulans was the most common fungus present. Apple pastries also displayed brand variation, but were unique in having many mesophilic aspergilli. This genus was generally absent from other products. The Penicillium content of apple pastries was also rather high; 50% of the psychrophilic flora was represented by this genus. The psychrophilic fungal flora of chicken pies was composed primarily of penicillia (50%) and Chrysosporium pannorum (46%).     

Effect of n-Alkanols on Acidification Curves of Aureobasidium pullulans Suspensions

Moragues, M. D., Estevez, J. J., Rementerı́a, A., and Sevilla, M. J. 1994. Effect of n-alkanols on acidification curves of Aureobasidium pullulans suspensions. Experimental Mycology 18, 1-6. n -Alkanols, from methanol to 1-hexanol, induce yeast-to-hyphae transition in the dimorphic fungus Aureobasidium pullulans. In order to elucidate whether triggering of the morphogenetic event is membrane related, we have studied the effect of the morphogenetic n-alkanols on the pH of suspensions of A. pullulans, with no external carbon source. n -Alkanols, at their hyphal inducing concentration or higher, caused a decrease in the initial acidification rate (C) of yeast-phase cell suspensions of A. pullulans. From this effect on C, an inhibition coefficient (K) was deduced, specific for each alcohol. These coefficients were directly related to the lipid/buffer partition coefficients of the alkanols. On the other hand, germ tubes of A. pullulans, obtained in the presence of n-propanol, showed a much slower acidification rate in water than yeast cells. Moreover, 1-propanol or 1-butanol did not significantly affect the initial acidification rate of germ tubes. The latter observation was interpreted as an adaptation of cells grown in the presence of alcohol. The results of all these experiments support our hypothesis that the plasma membrane is a target of the morphogenetic effects of n-alkanols.