Biological pretreatment and bioconversion of agricultural wastes,using ligninolytic and cellulolytic fungal consortia

Agricultural wastes have attractive potential as alternate energy sources. However, a major bottleneck is to identify eco-friendly treatment methodologies to utilize them. The large diversity of unexplored, novel, and potential microorganisms hold great promise and require periodic isolation and characterization of microorganisms for bioprospection. In this study, approximately 100 fungal isolates were tested for their lignocellulolytic enzyme activities, based on plate assay, followed by quantification of enzyme activity. From this, M2E (Inonotus tropicalis) and 2a (Cerrena unicolor) showed good growth and proficient ligninolytic activity; isolates GK1 (Chaetomium globosum) and GK2 (Chaetomium brasiliense) exhibited exceptional cellulolytic activity on lignocellulosic substrates such as rice straw and sugarcane bagasse. Consortia of the potential ligninolytic and cellulolytic isolates were set up to determine their ability to biodegrade the lignocellulosic substrates such as rice straw and sugarcane bagasse. The efficiency of the consortia was determined on the basis of the increase in enzyme activity; it was also evident through scanning electron microscopy, x-ray diffraction analysis of the degraded substrates, and the sugar yield. Experiments were also carried out to compare the biological with the physical pretreatment methods. The consortium of ligninolytic and cellulolytic marine-derived fungi developed in this study prove to have the potential for application in the effective utilization of agricultural wastes.     

Polysaccharide and protein degradation,germination, and virulence against mosquitoes in the entomopathogenic fungus Metarhizium anisopliae

From a genetically uniform wild-type strain of Metarhizium anisopliae pathogenic to mosquitoes, mutants were selected which were altered in the ability to degrade starch, gelatin, or milk. The mutants with enhanced starch degradation (dep), when grown on starch-containing media, proved hypervirulent toward the mosquito Culex pipiens pipiens in standard laboratory tests. Alterations in protein (gelatin or milk) degradation did not correlate with changes in virulence. The dep mutants appear to belong to the same class as mutants selected previously as hypervirulent and characterized by early spore germination. The relationship among polysaccharide degradation, early germination, and virulence is discussed.     

Acid production by Metarhizium anisopliae: Effects on virulence against mosquitoes and on detection of in vitro amylase,protease, and lipase activity

The imperfect fungus Metarhizium anisopliae infects and kills larvae of many insect species, including the mosquito Culex pipiens. Mutants of M. anisopliae selected for enhanced production of amylase have been found to have simultaneously acquired hypervirulence against C. pipiens larvae. In the present work, wild-type and some mutant strains of M. anisopliae were found to excrete an acid or acids which alter the pH of fungal cultures below that permissible for amylase activity. The amylase-enhanced hypervirulence mutants did not excrete acid. Detection of protease and lipase activities was complicated by the acid excretion. When this was taken into account, mutants having altered lipase or protease production were found to have no alteration in virulence against mosquito larvae. The link between acid excretion, amylase activity, and hypervirulence is discussed.     

Genomic analysis reveals cryptic diversity in aphelids and sheds light on the emergence of Fungi

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Changes in leaf gas exchange,chlorophyll a fluorescence and antioxidant metabolism within wheat leaves infected by Bipolaris sorokiniana

The photosynthetic performance (leaf gas exchange and chlorophyll a (Chla) fluorescence), activities of antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), ascorbate peroxidase (APX)] and the concentrations of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) in the flag leaves of plants from two wheat cultivars with contrasting levels of resistance to spot blotch was assessed. Spot blotch severity was significantly lower in plants from cv. BR‐18 compared to cv. Guamirim. Net carbon assimilation rate, stomatal conductance and concentrations of Chla, Chlab and carotenoids were significantly decreased from fungal infection. In contrast, internal CO₂ concentration was significantly increased from fungal infection in comparison to their non‐inoculated counterparts. Similarly, inoculation significantly reduced photochemical performance in the inoculated flag leaves in comparison to their non‐inoculated counterparts. However, plants from cv. BR‐18 were able to sustain greater functionality of the photosynthetic apparatus during fungal infection process compared to cv. Guamirim. The activities of SOD, POX, APX and CAT increased in inoculated flag leaves from both cultivars compared to non‐inoculated plants, and the highest increases were measured in cv. BR‐18. The greater activities of these enzymes were associated with a reduced H₂O₂ concentration in the inoculated flag leaves from cv. BR‐18, resulting, therefore, in a lower MDA concentration. Thus, a more efficient antioxidative system in flag leaves from cv. BR‐18 plays a pivotal role in removing the excess reactive oxygen species that were generated during the infection process of Bipolaris sorokiniana, therefore limiting cellular damage and largely preserving the photosynthetic efficiency of the infected flag leaves.     

Hydrolytic Activities of Fusarium solani and Fusarium solani f. sp. eumartii Associated with the Infection Process of Potato Tubers

The development of dry rot caused by Fusarium solani f. sp. eumartii was evaluated in susceptible (Huinkul) and resistant (Spunta) potato cultivars. Fungal proteolytic and polygalacturanase activities were measured at different days postinoculation either with the pathogenic F. solani f. sp. eumartii, isolate 3122 or with the non‐pathogenic F. solani, isolate 1042. After inoculation with the pathogenic fungus, proteolytic and polygalaturonase activities were higher in the susceptible than in the resistant cultivar. In addition, we found a correlation between the levels of proteolytic activity detected in the intercellular washing fluids with the size of the lesion area caused by F. solani f. sp. eumartii in Huinkul tubers. The action of the proteolytic activity over cell wall proteins of both potato cultivars was assayed. An extracellular potato protein with homology to proteinase inhibitors of the Kunitz family was identified as a substrate of the proteolytic activity in the susceptible cultivar. A microscopic study revealed differences between the potato genotypes in the rate of response to infection by F. solani f. sp. eumartii. In addition, the cell wall alteration caused by F. solani f. sp. eumartii in cortical cells of susceptible tubers was evaluated. The data with respect to the correlation between the course of cyto‐ and biochemical events of the two host–pathogen interactions were discussed.     

The fungi in Century 21. The XXI Fungal Genetics Conference, Pacific Grove, California, March 13-18, 2001

As an important opportunistic pulmonary pathogen, Pneumocystis carinii has been the focus of extensive research over the decades. The use of laboratory animal models has permitted a detailed understanding of the host-parasite interaction but an understanding of the basic biology of P. carinii has lagged due in large part to the inability of the organism to grow well in culture and to the lack of a tractable genetic system. Molecular techniques have demonstrated extensive heterogeneity among P. carinii organisms isolated from different host species. Characterization of the genes and genomes of the Pneumocystis family has supported the notion that the family comprises different species rather than strains within the genus Pneumocystis and contributed to the understanding of the pathophysiology of infection. Many of the technical obstacles in the study of the organisms have been overcome in the past decade and the pace of research into the basic biology of the organism has accelerated. Biochemical pathways have been inferred from the presence of key enzyme activities or gene sequences, and attempts to dissect cellular pathways have been initiated. The Pneumocystis genome project promises to be a rich source of information with regard to the functional activity of the organism and the presence of specific biochemical pathways. These advances in our understanding of the biology of this organism should provide for future studies leading to the control of this opportunistic pathogen.     

Lipid composition of the entomopathogenic fungus Nomuraea rileyi

The total lipid content, neutral and polar lipid composition, and fatty acid profiles were determined for the different developmental stages of the entomopathogenic fungus, Nomuraea rileyi. In general, the lipid composition of N. rileyi was found to be very similar to that of other Fungi Imperfecti. Triacylglycerides and sterols were the major neutral lipids and phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine were the major polar lipids. Phosphatidylcholine, resolved in conidial and mycelial samples, was not detected in hyphal body samples. The major fatty acids were palmitic, oleic, and linoleic. Stearic acid was detected as a minor component of hyphal body samples. During conidiogenesis the overall lipid composition was altered in (1) a reduction in total lipid content, (2) an increase in the polar/neutral lipid ratio, and (3) an increase in the degree of fatty acid unsaturation.     

真菌菌丝体和发酵液的氨基酸分析比较

<正> 对云芝等7种真菌的菌丝体和发酵液分别测定17种氨基酸的含量,测定结果表明这些菌丝体中含有丰富的人体8种必需氨基酸,支链氨基酸及精氨酸,而芳香族氨基酸和含硫氨基酸却很低。7种真菌的菌丝体和它们的子实体,和三种食用蛋白(α-酪蛋白,卵白蛋白,大豆球蛋白)及4种粮食产品     

Fungal evolution: major ecological adaptations and evolutionary transitions

Fungi are a highly diverse group of heterotrophic eukaryotes characterized by the absence of phagotrophy and the presence of a chitinous cell wall. While unicellular fungi are far from rare, part of the evolutionary success of the group resides in their ability to grow indefinitely as a cylindrical multinucleated cell (hypha). Armed with these morphological traits and with an extremely high metabolical diversity, fungi have conquered numerous ecological niches and have shaped a whole world of interactions with other living organisms. Herein we survey the main evolutionary and ecological processes that have guided fungal diversity. We will first review the ecology and evolution of the zoosporic lineages and the process of terrestrialization, as one of the major evolutionary transitions in this kingdom. Several plausible scenarios have been proposed for fungal terrestralization and we here propose a new scenario, which considers icy environments as a transitory niche between water and emerged land. We then focus on exploring the main ecological relationships of Fungi with other organisms (other fungi, protozoans, animals and plants), as well as the origin of adaptations to certain specialized ecological niches within the group (lichens, black fungi and yeasts). Throughout this review we use an evolutionary and comparative‐genomics perspective to understand fungal ecological diversity. Finally, we highlight the importance of genome‐enabled inferences to envision plausible narratives and scenarios for important transitions.     

Cellobiohydrolase II is the main conidial-bound cellulase in Trichoderma reesei and other Trichoderma strains

Monoclonal antibodies have been used to determine the presence of cellobiohydrolases I and II (CBH I and II), and endoglucanase I (EG I) on the surface of conidia from Trichoderma reesei QM 9414 and RUT C-30, and 8 other Trichoderma species. For this purpose, proteins were released from the conidial surface by treatment with a non-ionic detergent (Triton X-100 and -octylglucoside), followed by SDS-PAGE/Western blotting and immunostaining. Both CBH I and II were clearly present, but — unlike in extracellular culture fluids from Trichoderma — CBH II was the predominant cellulase. In T. reesei EG I could not be detected. The higher producer strain T. reesei RUT C-30 exhibited a higher conidial level of CBH II than T. reesei QM 9414. In order to assess the importance of the conidial CBH II level for cellulase induction by cellulose, multiple copies of the chb2 gene were introduced into the T. reesei genome by cotransformation using PyrG as a marker. Stable multicopy transformants secreted the 2- to 4-fold level of CBH II into the culture medium when grown on lactose as a carbon source, but their CBH I secretion was unaltered. Upon growth on cellulose, both CBH I and CBH II secretion was enhanced. Those strain showing highest cellulase activity on cellulose also appeared to contain the highest level of conidial bound CBH II. CBH II was also the predominant conidial cellulase in various other Trichoderma sp. However, roughly the same amount of conidial bound CBH II was detected in all strains, although their cellulase production differed considerably.     

Histochemical detection of laccase in Pycnoporus cinnabarinus using microwave-enhanced colloidal gold microcrystallization

Laccase was detected histochemically in Pycnoporus cinnabarinus fungal hyphae by growing the organism on cellophane or microporous polycarbonate membranes overlaid on malt extract agar. Membrane sections were stained with a reagent of 2,6-dimethoxyphenol and colloidal gold for 30 min and irradiated for 24 s at maximum output in a microwave oven. Laccase was detected as purple-blue deposits at the apical tip region in high concentrations, and at lower levels on the exterior, sub-apical hyphal sheath.