Hyphal morphogenesis in Aspergillus nidulans

The formation of hyphae that grow solely by apical extension is a defining feature of filamentous fungi. Hyphal morphogenesis involves several key steps, including the establishment and maintenance of a stable polarity axis, as well as cell division via the deposition of septa. Several filamentous fungi have been employed in attempts to decipher the mechanisms underlying these steps. Amongst these fungi, Aspergillus nidulans has proven to be a particularly valuable model. The genetic tractability of this fungus coupled with the availability of sophisticated post-genomics resources has enabled the identification and characterization of numerous genes involved in hyphal morphogenesis. Here, we summarize current progress towards understanding the function of these genes and the mechanisms involved in polarized hyphal growth and septation in A. nidulans. We also highlight important areas for future investigation.     

Efficient transformation and expression of Vitreoscilla haemoglobin in the biological control bacterium Collimonas pratensis ZL261

Collimonas species are soil bacteria characterised by their ability to attach to and utilise fungi as a food source (mycophagy), as well as their chitin-degrading capacity (via chitinase production). These attributes, alongside volatile compounds, are thought to contribute to their function as fungal antagonists, including economically important plant pathogens. Despite this, studies have found no relationship between antifungal activity and chitinase production, or volatile compounds in Collimonas pratensis isolate ZL261, and there have been no studies on genetic control and regulatory biosynthesis of antifungal substances in Collimonas species. In this study, we showed that low concentrations of dissolved oxygen were unfavourable for growth and antifungal activity. We successfully introduced the gene vgb encoding Vitreoscilla haemoglobin (VHb) into isolate ZL261. The heterologous expression of VHb not only enhanced cell growth, but also improved antifungal activity against the brown rot fungus Monilinia fructicola under oxygen-restricted conditions; 18.6% of untreated peach fruits were infected (average lesion diameter: 9.2?mm), while only 10.8% of fruit treated with the transformed isolate, ZV261, were infected (average lesion diameter: 5.4?mm). These results suggest that the antagonism have been due to the secreted secondary metabolites, which are sensitive to the oxygen-restricted conditions.     

Antifungal potential of extracellular metabolites produced by Streptomyces hygroscopicus against phytopathogenic fungi

Indigenous actinomycetes isolated from rhizosphere soils were assessed for in vitro antagonism against Colletotrichum gloeosporioides and Sclerotium rolfsii. A potent antagonist against both plant pathogenic fungi, designated SRA14, was selected and identified as Streptomyces hygroscopicus. The strain SRA14 highly produced extracellular chitinase and β-1,3-glucanase during the exponential and late exponential phases, respectively. Culture filtrates collected from the exponential and stationary phases inhibited the growth of both the fungi tested, indicating that growth suppression was due to extracellular antifungal metabolites present in culture filtrates. The percentage of growth inhibition by the stationary culture filtrate was significantly higher than that of exponential culture filtrate. Morphological changes such as hyphal swelling and abnormal shapes were observed in fungi grown on potato dextrose agar that contained the culture filtrates. However, the antifungal activity of exponential culture filtrates against both the experimental fungi was significantly reduced after boiling or treatment with proteinase K. There was no significant decrease in the percentage of fungal growth inhibition by the stationary culture filtrate that was treated as above. These data indicated that the antifungal potential of the exponential culture filtrate was mainly due to the presence of extracellular chitinase enzyme, whereas the antifungal activity of the stationary culture filtrate involved the action of unknown thermostable antifungal compound(s).     

Genomic analysis of the aconidial and high-performance protein producer,industrially relevant Aspergillus niger SH2 strain

Aspergillus niger is usually regarded as a beneficial species widely used in biotechnological industry. Obtaining the genome sequence of the widely used aconidial A. niger SH2 strain is of great importance to understand its unusual production capability. In this study we assembled a high-quality genome sequence of A. niger SH2 with approximately 11,517 ORFs. Relatively high proportion of genes enriched for protein expression related FunCat items verify its efficient capacity in protein production. Furthermore, genome-wide comparative analysis between A. niger SH2 and CBS513.88 reveals insights into unique properties of A. niger SH2. A. niger SH2 lacks the gene related with the initiation of asexual sporulation (PrpA), leading to its distinct aconidial phenotype. Frame shift mutations and non-synonymous SNPs in genes of cell wall integrity signaling, β-1,3-glucan synthesis and chitin synthesis influence its cell wall development which is important for its hyphal fragmentation during industrial high-efficiency protein production.     

Bacterial farming by the fungus Morchella crassipes

The interactions between bacteria and fungi, the main actors of the soil microbiome, remain poorly studied. Here, we show that the saprotrophic and ectomycorrhizal soil fungus Morchella crassipes acts as a bacterial farmer of Pseudomonas putida, which serves as a model soil bacterium. Farming by M. crassipes consists of bacterial dispersal, bacterial rearing with fungal exudates, as well as harvesting and translocation of bacterial carbon. The different phases were confirmed experimentally using cell counting and ¹³C probing. Common criteria met by other non-human farming systems are also valid for M. crassipes farming, including habitual planting, cultivation and harvesting. Specific traits include delocalization of food production and consumption and separation of roles in the colony (source versus sink areas), which are also found in human agriculture. Our study evidences a hitherto unknown mutualistic association in which bacteria gain through dispersal and rearing, while the fungus gains through the harvesting of an additional carbon source and increased stress resistance of the mycelium. This type of interaction between fungi and bacteria may play a key role in soils.     

Mycelial carton galleries of Azteca brevis (Formicidae) as a multi-species network

Apart from growing fungi for nutrition, as seen in the New World Attini, ants cultivate fungi for reinforcement of the walls of their nests or tunnel-shaped runway galleries. These fungi are grown on organic material such as bark, epiphylls or trichomes, and form stable ‘carton structures’. In this study, the carton of the runway galleries built by Azteca brevis (Formicidae, Dolichoderinae) on branches of Tetrathylacium macrophyllum (Flacourtiaceae) is investigated. For the first time, molecular tools are used to address the biodiversity and phylogenetic affinities of fungi involved in tropical ant carton architecture, a previously neglected ant–fungus mutualism.The A. brevis carton involves a complex association of several fungi. All the isolated fungi were unequivocally placed within the Chaetothyriales by DNA sequence data. Whereas five types of fungal hyphae were morphologically distinguishable, our DNA data showed that more species are involved, applying a phylogenetic species concept based on DNA phylogenies and hyphal morphology. In contrast to the New World Attini with their many-to-one (different ant species—one fungal cultivar) pattern, and temperate Lasius with a one-to-two (one ant species—two mutualists) or many-to-one (different ant species share the same mutualist) system, the A. brevis–fungi association is a one-to-many multi-species network. Vertical fungus transmission has not yet been found, indicating that the A. brevis–fungi interaction is rather generalized.     

Design, synthesis, and evaluations of antifungal activity of novel phenyl(2H-tetrazol-5-yl)methanamine derivatives

Fungal infections pose a continuous and serious threat to human health and life. The intrinsic resistance has been observed in many genera of fungi. Many fungal infections are caused by opportunistic pathogens that may be endogenous (Candida infections) or acquired from the environment (Cryptococcus and Aspergillus infections). So, new therapeutic strategies are needed to combat various fungal infections. Fluconazole shows good antifungal activity with relatively low toxicity and is preferred as first line antifungal therapy, but it has suffered from severe drug resistance. So, there is a need to design novel analogues by modification of fluconazole-like structure. A novel series of phenyl(2H-tetrazol-5-yl)methanamine derivatives were synthesized by reaction of α-amino nitrile with sodium azide and ZnCl₂ in presence of isopropyl alcohol. They were evaluated for antifungal activity against Candida albicans and Aspergillus niger and subjected to docking study against 1EA1.     

Interactions between fungi and bacteria influence microbial community structure in the Megachile rotundata larval gut

Recent declines in bee populations coupled with advances in DNA-sequencing technology have sparked a renaissance in studies of bee-associated microbes. Megachile rotundata is an important field crop pollinator, but is stricken by chalkbrood, a disease caused by the fungus Ascosphaera aggregata. To test the hypothesis that some gut microbes directly or indirectly affect the growth of others, we applied four treatments to the pollen provisions of M. rotundata eggs and young larvae: antibacterials, antifungals, A. aggregata spores and a no-treatment control. We allowed the larvae to develop, and then used 454 pyrosequencing and quantitative PCR (for A. aggregata) to investigate fungal and bacterial communities in the larval gut. Antifungals lowered A. aggregata abundance but increased the diversity of surviving fungi. This suggests that A. aggregata inhibits the growth of other fungi in the gut through chemical or competitive interaction. Bacterial richness decreased under the antifungal treatment, suggesting that changes in the fungal community caused changes in the bacterial community. We found no evidence that bacteria affect fungal communities. Lactobacillus kunkeei clade bacteria were common members of the larval gut microbiota and exhibited antibiotic resistance. Further research is needed to determine the effect of gut microbes on M. rotundata health.     

温郁金内生真菌Chaetomium globosum L18对植物病原菌的抑菌谱及拮抗机理

通过体外培养法,研究了药用植物温郁金内生真菌Chaetomium globosum L18对几种常见的植物病原菌的抑菌谱及其拮抗机理。结果表明,Chaetomium globosum L18对多种植物病原真菌和细菌均有不同程度的抑制作用,具有较广的抑菌谱,但对不同植物病原菌的抑制作用具有显著性差异(P<0.05),抑制率最高可达到92.9%;抑菌机制结果显示,竞争作用和重寄生作用是其主要的拮抗机制之一;发酵产物抑制作用测定发现,内生真菌Chaetomium globosum L18能够分泌产生抗菌物质抑制病原菌菌丝的生长和孢子萌发,可引起病原菌菌丝菌丝膨大成串珠状,分枝增多,分枝顶端膨胀后细胞壁破裂,原生质外溢,产生溶菌作用;使分生孢子萌发畸形,萌发率降低。     

Silver nanoparticle production by the fungus Fusarium oxysporum: nanoparticle characterisation and analysis of antifungal activity against pathogenic yeasts

The microbial synthesis of nanoparticles is a green chemistry approach that combines nanotechnology and microbial biotechnology. The aim of this study was to obtain silver nanoparticles (SNPs) using aqueous extract from the filamentous fungus Fusarium oxysporum as an alternative to chemical procedures and to evaluate its antifungal activity. SNPs production increased in a concentration-dependent way up to 1 mM silver nitrate until 30 days of reaction. Monodispersed and spherical SNPs were predominantly produced. After 60 days, it was possible to observe degenerated SNPs with in additional needle morphology. The SNPs showed a high antifungal activity against Candida and Cryptococcus , with minimum inhibitory concentration values ≤ 1.68 µg/mL for both genera. Morphological alterations of Cryptococcus neoformans treated with SNPs were observed such as disruption of the cell wall and cytoplasmic membrane and lost of the cytoplasm content. This work revealed that SNPs can be easily produced by F. oxysporum aqueous extracts and may be a feasible, low-cost, environmentally friendly method for generating stable and uniformly sized SNPs. Finally, we have demonstrated that these SNPs are active against pathogenic fungi, such as Candida and Cryptococcus .     

Metabolites of rhizobacteria antagonistic towards fungal plant pathogens

Biological control of insect, plant pathogens and weeds is the only major alternative to the use of pesticides in agriculture and forestry. A double-layer technique was used for isolation of antagonistic bacteria from rhizosphere against plant pathogenic fungi. Four potential rhizobacteria was selected in dual culture plate method based on their antifungal activity against several soil-borne fungal plant pathogens. The selected rhizobacteria, identified based on their morphological, biochemical and molecular traits, belong to the species of fluorescentPseudomonas (SAB8, GM4) andBacillus (A555, GF23). The active antifungal metabolites produced by these strains in culture filtrates were tested for the growth inhibition ofFusarium semitectum used as test fungus. The active fraction of antifungal metabolite/(s) from fluorescentPseudomonas (SAB8, GM4) and their effects on hyphal growth were observed under microscope. Two kinds of alterations were detected: inhibition of hyphal tip elongation and an extensive branching of hyphae with closer septa.     

New trifluoromethyl quinolone derivatives: Synthesis and investigation of antimicrobial properties

A series of quinolone derivatives, containing different heterocyclic amines were prepared. Synthesized compounds were evaluated for their in vitro antimicrobial activities against two Gram-positive bacteria, three Gram-negative bacteria as well as four fungi. All the derivatives showed good activity towards Gram-positive bacteria and less activity towards Gram-negative bacteria. They also showed moderate to comparable activity against Aspergillus niger and Candida albicans and low to moderate antifungal activity against Aspergillus fumigatus and Aspergillus flavus.     

Muscodor albus MOW12 an Endophyte of Piper nigrum L. (Piperaceae) Collected from North East India Produces Volatile Antimicrobials

Muscodor albus MOW12, an endophytic fungus isolated from Piper nigrum in Mawlong, Meghalaya, India, resembles some cultural and hyphal characteristics of previous isolates of Muscodor sp. In addition, it possesses about 99 % similarity in its ITS rDNA with other M. albus isolates and thus is nicely centered within the genetic tree to other Muscodor spp. This xylariaceae fungus effectively inhibits and kills certain plant pathogenic fungi by virtue of a mixture of volatile compounds that it produces. The majority of these compounds were identified by gas chromatography/mass spectrometry as small molecular weight esters, alcohols, and acids. The main ester components of this isolate of M. albus in its volatile mixture are acetic acid, ethyl ester; propanoic acid, 2-methyl-, methyl ester and acetic acid, 2-methylpropyl ester. This appears to be the first report of any M. albus strain from India.     

Diverse and bioactive endophytic Aspergilli inhabit Cupressaceae plant family

Aspergilli are filamentous, cosmopolitan and ubiquitous fungi which have significant impact on human, animal and plant welfare worldwide. Due to their extraordinary metabolic diversity, Aspergillus species are used in biotechnology for the production of a vast array of biomolecules. However, little is known about Aspergillus species that are able to adapt an endophytic lifestyle in Cupressaceae plant family and are capable of producing cytotoxic, antifungal and antibacterial metabolites. In this work, we report a possible ecological niche for pathogenic fungi such as Aspergillus fumigatus and Aspergillus flavus. Indeed, our findings indicate that A. fumigatus, A. flavus, Aspergillus niger var. niger and A. niger var. awamori adapt an endophytic lifestyle inside the Cupressaceous plants including Cupressus arizonica, Cupressus sempervirens var. fastigiata, Cupressus semipervirens var. cereiformis, and Thuja orientalis. In addition, we found that extracts of endophytic Aspergilli showed significant growth inhibition and cytotoxicity against the model fungus Pyricularia oryzae and bacteria such as Bacillus sp., Erwinia amylovora and Pseudomonas syringae. These endophytic Aspergilli also showed in vitro antifungal effects on the cypress fungal phytopathogens including Diplodia seriata, Phaeobotryon cupressi and Spencermartinsia viticola. In conclusion, our findings clearly support the endophytic association of Aspergilli with Cupressaceae plants and their possible role in protection of host plants against biotic stresses. Observed bioactivities of such endophytic Aspergilli may represent a significant potential for bioindustry and biocontrol applications.     

Mathematical modelling and assessment of the pH homeostasis mechanisms in Aspergillus niger while in citric acid producing conditions

In this work we introduce an extended model of the Aspergillus niger metabolism while in citrate production conditions. The model includes many recent findings related to various transport processes. It now considers a new information about the fructose uptake system and the proton and amino acids carriers between cytoplasm and the external medium. It also accounts for recent information about both the malate-citrate antiport between mitochondria and cytoplasm and the dihydrogen citrate ion excretion symport with protons. Finally, the model also accounts for new information about the glycerol-3-phosphate shuttle and pH buffering systems. Provided with this updated representation and after having assessed its quality and dynamic behaviour, we were able to explain the observed pH homoeostasis found in A. niger while in citrate producing conditions. The model also serves to enhance our comprehension of the molecular mechanisms operating in order to keep homoeostasis of pH in A. niger and other fungi, bacteria and yeast of biotechnological relevance.