Advancing Frontiers in Mycology & Mycotechnology: Basic and Applied Aspects of Fungi

685 Pages·2019·17.399 MB·English

by  Tulasi Satyanarayana, Sunil Kumar Deshmukh, Mukund V. Deshpande

Tulasi Satyanarayana 
Sunil Kumar Deshmukh 
Mukund V. Deshpande Editors
Advancing
Frontiers in
Mycology &
Mycotechnology
Basic and Applied Aspects of Fungi
Advancing Frontiers in Mycology
& Mycotechnology
Tulasi Satyanarayana
Sunil Kumar Deshmukh
Mukund V. Deshpande
Editors
Advancing Frontiers
in Mycology
& Mycotechnology
Basic and Applied Aspects of Fungi
Editors
Tulasi Satyanarayana Sunil Kumar Deshmukh
Division of Biological Science and Biotech & Management of
Engineering Bioresources Div
Netaji Subhas University of Technology The Energy and Resources Institute
New Delhi, Delhi, India New Delhi, Delhi, India
Mukund V. Deshpande
Division of Biological Sciences
CSIR-National Chemical Laboratory
Pune, Maharashtra, India
ISBN 978-981-13-9348-8 ISBN 978-981-13-9349-5 (eBook)
https://doi.org/10.1007/978-981-13-9349-5
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Foreword
The term ‘mycology’ dates back to the eighteenth century, and in its formative
years, the study of fungi was guided by reputed botanists like C. H. Persoon,
E.M. Fries and A. deBary. During the nineteenth and twentieth centuries, phenom-
enal progress in classical mycology took place, including descriptive morphology,
identification, classification and taxonomy of fungi primarily, due to the efforts of
botanists, despite the fact that fungi are not phylogenetically related to plants. The
reason for this essentially stems from the devastating fungal diseases in plants, nota-
bly the crop plants, causing serious problems in agriculture. There was an urgent
need to understand the aetiology of the destructive fungal diseases and devise suit-
able control measures to save the crops. As an example may be mentioned the stud-
ies in India by Prof. M.J. Narasimhan (1930) who collected and studied diverse
heterothallic strains of the destructive Phytophthora1 infection on areca nut palms
and devising fungicidal treatment schedules for controlling the disease in the rain-
forest areas of Karnataka (Mysore) state.
During the early years, traditional mycological knowledge facilitated the (a)
development of various oriental fermented foods, such as miso, tempeh and sake,
based on strains of fungi such as Aspergillus oryzae, Rhizopus oligosporus and oth-
ers (Hesseltine 1983), (b) differentiation of edible and poisonous mushrooms and
(c) understanding the aetiology of ergot poisoning that caused serious convulsions
due to consumption of the ergot sclerotia contaminating the grain which is con-
sumed as food.
During the twentieth century, several milestone advances in biological sciences
based on mycological studies can be recognized. As examples, we may mention the
following:
(a) The Nobel Prize-winning (1958) studies on Neurospora by G.W. Beadle and
E.L. Tatum which resulted in their discovery of how genes act by regulating
definite chemical events that in turn affected the development in organisms.
Based on ingenious selection of mutants deficient in the synthesis of single
growth factors, Beadle and Tatum concluded that mutations to genes affected
1 Recently, the genera Phytophthora and Pythium have been placed in Kingdom Chromista or
Kingdom Straminipila, distinct from Kingdom Fungi (Ho 2018).
v
vi Foreword
the enzymes of organisms. It is noteworthy that these studies provided the early
link between genetics and the upcoming new scientific field of molecular
biology.
(b) The discovery of truly anaerobic chytrids present in the rumen of herbivores
and playing a significant role in the digestion of the cellulosic feed is a mile-
stone discovery of the twentieth century. Classified under Neocallimastigaceae,
these chytrids with chitinous cell walls are truly anaerobes lacking mitochon-
dria, cytochromes and other biochemical features of the oxidative phosphoryla-
tion pathway. The group possesses organelles (hydrogenosomes) for a major
part of anaerobic energy metabolism (Nagpal et al. 2009).
Taxonomy and classification of fungi have been central to the mycological stud-
ies, and this has led to a well-established database for understanding fungal biodi-
versity. Classical taxonomy relied upon the morphological features of the spore
forms. With the advent of biochemical and molecular data becoming available, sev-
eral conceptual changes in fungal taxonomy have arisen. While giving an opportu-
nity to gain newer knowledge, these developments also pose several challenges and
opinion divides in their interpretation and implementation.
Classical taxonomy classified all the zoospore-forming fungi under Phycomycetes
considering their monophyletic origin from algae. Recent advances in biochemical
knowledge have established that the Chytridiomycetes2 with chitinous cell walls,
despite being zoospore-forming, are distinct from the rest of the zoospore-forming
fungi with β-glucan in their cell walls, and these are grouped together under the
Oomycota (as opposed to the Eomycota under which the Zygomycetes, Ascomycetes
and Basidiomycetes are classified). The term Phycomycetes as originally adapted is
no longer valid and, hence, abandoned from present-day mycological literature.
DNA sequencing and bar-coding of fungal species are major developments
which offer challenges to accepted morphology-based taxonomy. The nuclear ribo-
somal internal transcribed spacer (ITS) is widely used as a DNA bar-coding marker
to characterise the diversity and composition of fungal communities. Since the
1990s, the ITS region has been extensively used in molecular methods as well as
ecological studies on fungi.
Estimates of fungal biodiversity have undergone sharp changes following envi-
ronmental DNA sequencing of fungal ITS reads. Currently, there are around 81,000
accepted fungal names out of a total of up to 5.1 million species (Blackwell 2011).
Environmental DNA sequencing has accumulated more than one million fungal ITS
reads. Whether ‘sequence-based voucher-less fungi’ (in the absence of actual speci-
mens) should be given a formal nomenclature is widely debated, and the opinions
of expert mycologists are sharply divided on this point with reference to the estima-
tion of biodiversity of fungi in the natural environment. Several informative reviews
on DNA bar-coding as well as sequence-based fungal nomenclature are published
and may be consulted for a more complete understanding of the problem and its
2 Now chytrids are grouped under Chytridiomycota (N. P. Money, in The Fungi (Third Edition),
2016)
Foreword vii
perspectives (Shenoy et al. 2007; Begerow et al. 2010; Das and Deb 2015; Hibbett
et al. 2016; Hongsanen et al. 2018).
Conventionally, taxonomic principles related to fungi are debated and decisions
taken under the authority of the International Code of Botanical Nomenclature
(ICBN) held once in 6 years during the International Botanical Congress. Recently,
the ICBN has been renamed as International Code of Nomenclature for algae, fungi
and plants. In the Melbourne ICBN held in 2011, a momentous decision was taken
to abolish Article 59 which permitted the use of dual nomenclature for pleomorphic
fungi. In the morphology-based taxonomy, anamorphs (asexual forms) and teleo-
morphs (sexual forms) of fungi discovered and described independently could
receive different but valid names as ruled by Article 59 of the ICBN. In the present
era of phylogenetic molecular analysis, co-ordination of anamorphic and teleomor-
phic elements of a fungus can, in principle, be unequivocally established. Molecular
mycologists are strongly in favour of the unification of the fungal nomenclature and
achieve the ‘one fungus-one name’ ideal. However, there are several practical hur-
dles, and several specialist committees are in serious debates to elucidate the practi-
cal feasibility of implementing the same (Gams 2014).
Simultaneous with the focused attention on fungi as troublesome pathogens of
plants, animals and humans, beneficial attributes of fungi as sources of valuable
metabolites were also realized. Taka-Diastase from A. oryzae and citric acid from
Aspergillus niger are examples of fungal-based processes of manufacture in the late
nineteenth and early twentieth centuries. With the discovery of penicillin and the
dawn of the antibiotic era, the decades of the twentieth century became an era for
screening diverse microorganisms including fungi for discovering novel bioactive
molecules and producing them by fermentation. Several industrially useful enzymes,
hormones, vitamins and growth factors of fungal origin spearheaded the fermenta-
tion technology development. Fungi and their metabolites were also employed in
the biotransformation of organic molecules (e.g. steroids).
The beneficial attributes of fungi far outweighed their earlier negative image, and
this led to a very healthy and positive attitude towards fungi which deserved to be
studied for deriving beneficial products from their metabolism. The term ‘biodiver-
sity’ acquired a newer dimension in terms of its relevance to applied microbiologi-
cal and biotechnological research. An immense focus on the physiological,
biochemical and molecular aspects of mycodiversity became imminent for biopro-
cess development based on novel fungal strains.
Heterologous gene expression and production of mammalian proteins in fungal
hosts have been successfully carried out for chymosin (cheese manufacture enzyme)
in A. niger var. awamori and insulin in Komagataella (Pichia) pastoris.
Spectacular advances in both mycology and mycotechnology during the twenti-
eth century have added new dimensions to mycological research, necessitating
newer approaches to achieve success in this field at present. A different mindset and
training have become necessary in order to carry out meaningful research projects
viii Foreword
in the twenty-first century. Some of the salient features of this new preparation may
be defined as follows:
(a) Knowledge-based exploration of fungal biodiversity in different ecosystems by
mycologists is an important aspect. Understanding the type of habitats in which
specific types of fungal populations would be naturally enriched will be very
helpful. For example, screening for thermophiles in self-heating composts,
bagasse piles or sun-heated soils would be very useful. The utilization of special
traits in specific groups of fungi is also very beneficial, such as the overlaying
of agar plates with moistened detritus to allow colonies to develop from forcibly
‘shot’ spores in the case of saprophytic Entomophthorales like Conidiobolus
and Basidiobolus as well as yeasts like Sporobolomyces. Adopting selective iso-
lation techniques including the use of specific inhibitory chemicals or antibiot-
ics has significantly enabled the slower growing and rarer populations to be
isolated on agar plates overcoming competition from the rapidly growing fungal
populations. The topic of selective isolation is a vast one and has been reviewed
(Srinivasan 2004, 2008) which may be consulted.
(b) Apart from taxonomic studies, in this biotechnology era, mycologists have the
responsibility of conserving their discoveries in pure culture through optimiza-
tion of culture preservation methods suited for their specific strains. They have
to ensure both morphological and genetic stability of their cultures under the
laboratory conditions for prolonged periods. Repeated and frequent subcultures
on sugar-rich media, like potato dextrose agar, often adversely affect sporula-
tion, particularly in strains associated with decomposing plant litter. While
freeze-drying (lyophilisation) is widely accepted as a safe method of long-term
conservation, it must be verified in regard to its suitability for the strains under
study. It is experienced that some fungi (e.g. Conidiobolus) failed to survive
lyophilisation, while many of the lignocellulose-degrading hyphomycetes
showed reduced sporulation upon revival from lyophilized cultures. After in-
depth studies to standardize the optimum conservation method(s), mycologists
in India should deposit their cultures in the national fungal germplasm banks
such as the National Fungal Culture Collection of India (NFCCI), Agharkar
Research Institute, Pune; Microbial Type Culture Collection (MTCC), CSIR-
Institute of Microbial Technology, Chandigarh; National Centre for Microbial
Resource (NCMR), National Centre for Cell Science, Pune; or the National
Collection of Industrial Microorganisms (NCIM), CSIR-National Chemical
Laboratory, Pune. While depositing the cultures, it is mandatory that the full
information on the optimal methods of conservation for the strain(s) is given to
the curators of the culture collections to follow for successful conservation.
(c) When exploring fungal diversity for novel metabolites, it may be necessary to
pay attention right from the isolation stage, keeping in mind the purpose of the
strain screening. I shall illustrate this point taking the case of ‘endophytic fungi’
and exploration of their potential to produce the plant metabolite by fermenta-
tion. Widespread interest in exploring endophyte fungal populations of diverse
medicinal plants has arisen after the report from Prof. Gary Strobel’s laboratory
Foreword ix
in the USA that an endophyte designated Taxomyces andreanae produced the
valuable anticancer metabolite taxol by fermentation (Stierle et al. 1993).
Presently, diverse endophytic fungi have been isolated and maintained in pure
culture in several laboratories with the purpose of studying their potential to
produce the valuable plant metabolites by fermentation. A point that deserves to
be seriously given consideration is as follows:
The endophytic fungus has imbibed the genes from the medicinal plant through
constant association. In the laboratory, culturing it on routine sugar-rich media
while facilitating satisfactory growth, there is no selection pressure for the growing
fungus to conserve these imbibed genes, and over several subcultures on routine
media may even be lost. An alternate suggestion which may be worth considering is
to plan the culture isolation of the endophyte on a relatively simple nutrient medium
fortified with some of the key chemical intermediate components involved in the
biosynthesis of the specific plant metabolite. It is possible on such a medium, the
metabolic genes for the plant metabolite are conserved better, and these may be
subjected to mutation and gene amplification through molecular techniques, leading
to strains which will be able to produce the metabolite at commercially viable lev-
els. Presently, the levels of production observed are very low and insignificant, and
much more studies are warranted before practical and viable technologies for
endophyte-b ased plant metabolites would become a reality.
Recent years have witnessed increased emphasis on the beneficial attributes of
fungi, recognizing their role in the natural ecosystems and as the source of several
metabolites of value for humankind. Rambold et al. (2013) in an article, suggesting
that mycology should be recognized as a major field in biology, stated ‘Given the
ecological and economic relevance of fungi...Mycology is insufficiently recognized
as a major field of science’. In the theme for the International Mycological Congress
held at Puerto Rico in July 2018, mycological discoveries for a better world and
how fungi contributed to the health of society and of ecosystem were given empha-
sis. Clearly, one can perceive the changing face of mycology from ‘fungi as destruc-
tive agents to be despised to fungi as true benefactors of humanity to be adored’.
In order to harness the full potential of fungi for mankind’s benefit, mycologists
must identify themselves as part of multidisciplinary teams exploring fungi for
novel metabolites. They should broaden their horizons of interest and acquire
knowledge in physiology, biochemistry and molecular biology of fungi. Familiarity
with natural product chemistry and bioprocess engineering for mould metabolites
including mould morphogenesis in fermentors related to growth conditions for
maximum yields are desirable.
Multidisciplinary collaboration to achieve success in technology is a must, and
the status of mycology in the twenty-first century is meaningfully reflected in the
following quote from Kreger (2003):
We all realise that most scientists in the future will be part of multi-disciplinary research
teams…the shift is causing a way scientists need to train … although they must still be an
expert in their specialty, they must also become conversant with techniques that once
seemed beyond their domain … they also need to recognize where their knowledge ends and
where they should seek the help from others….
x Foreword
It gives me pleasure in writing the foreword for this book, Advancing Frontiers
in Mycology and Mycotechnology: Basic and Applied Aspects of Fungi. The invited
chapters included in this book cover most of the aspects of fungal biology and
applied aspects of fungi. I wish to congratulate the editors and all the contributors
for their efforts in bringing out this book. I sincerely hope and wish that the book
will be useful for students, scholars, scientists and teachers of biology, microbiol-
ogy and biotechnology.
Ex-Head, Biochemical Sciences Division M. C. Srinivasan
CSIR-NCL
Pune, Maharashtra, India
R.H.17, Planet Millennium
Pune, Maharashtra, India
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