# The amazing potential of fungi: 50 ways we can exploit fungi industrially

Kevin Hyde, Jianchu Xu, Sylvie Rapior, Rajesh Jeewon, Saisamorn Lumyong, Allen Grace T. Niego, Pranami Abeywickrama, Janith Aluthmuhandiram, Rashika Brahamanage, Siraprapa Brooks, et al.

► To cite this version:

Kevin Hyde, Jianchu Xu, Sylvie Rapior, Rajesh Jeewon, Saisamorn Lumyong, et al. The amazing potential of fungi: 50 ways we can exploit fungi industrially. Fungal Diversity, 2019, 97 (1), pp.1-136. 10.1007/s13225-019-00430-9. hal-02196145

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# The amazing potential of fungi: 50 ways we can exploit fungi industrially

Kevin D. Hyde<sup>1,2,3,4,5,9</sup> · Jianchu Xu<sup>1,10,21</sup> · Sylvie Rapior<sup>22</sup> · Rajesh Jeewon<sup>18</sup> · Saisamorn Lumyong<sup>9,13</sup> ·

Allen Grace T. Niego<sup>2,3,20</sup> · Pranami D. Abeywickrama<sup>2,3,7</sup> · Janith V. S. Aluthmuhandiram<sup>2,3,7</sup> ·

Rashika S. Brahamanage<sup>2,3,7</sup> · Siraprapa Brooks<sup>3</sup> · Amornrat Chaiyasen<sup>28</sup> · K. W. Thilini Chethana<sup>2,3,7</sup> ·

Putarak Chomnunti<sup>2,3</sup> · Clara Chepkirui<sup>12</sup> · Boontiya Chuankid<sup>2,3</sup> · Nimali I. de Silva<sup>1,2,4,13</sup> ·

Mingkwan Doilom<sup>1,4,13</sup> · Craig Faulds<sup>6</sup> · Eleni Gentekaki<sup>3</sup> · Venkat Gopalan<sup>14</sup> · Pattana Kakumyan<sup>2,3</sup> ·

Dulanjalee Harishchandra<sup>2,3,7</sup> · Hridya Hemachandran<sup>24</sup> · Sinang Hongsanan<sup>26,27</sup> · Anuruddha Karunarathna<sup>2,17</sup> ·

Samantha C. Karunarathna<sup>1</sup> · Sehroon Khan<sup>10</sup> · Jaturong Kumla<sup>13,9</sup> · Ruvishika S. Jayawardena<sup>2,3</sup> ·

Jian-Kui Liu<sup>11</sup> · Ningguo Liu<sup>2,3</sup> · Thatsanee Luangharn<sup>1,21,22,29</sup> · Allan Patrick G. Macabeo<sup>12,23</sup> ·

Diana S. Marasinghe<sup>2,3</sup> · Dan Meeks<sup>19</sup> · Peter E. Mortimer<sup>1,10</sup> · Peter Mueller<sup>19</sup> · Sadia Nadir<sup>10,15,21</sup> ·

Karaba N. Nataraja<sup>16</sup> · Sureeporn Nontachaiyapoom<sup>3</sup> · Meghan O'Brien<sup>19</sup> · Watsana Penkhrue<sup>9,13</sup> ·

Chayanard Phukhamsakda<sup>2,3</sup> · Uma Shaanker Ramanan<sup>16,25</sup> · Achala R. Rathnayaka<sup>2,3</sup> · Resurreccion B. Sadaba<sup>29</sup> ·

Birthe Sandargo<sup>12</sup> · Binu C. Samarakoon<sup>2,3</sup> · Danushka S. Tennakoon<sup>2,3</sup> · Ramamoorthy Siva<sup>24</sup> ·

Wasan Sriprom<sup>9,13</sup> · T. S. Suryanarayanan<sup>30</sup> · Kanaporn Sujarit<sup>9,13</sup> · Nakarin Suwannarach<sup>9,13</sup> ·

Thitipone Suwunwong<sup>3,8</sup> · Benjarong Thongbai<sup>12</sup> · Naritsada Thongklang<sup>2</sup> · Deping Wei<sup>1,2,3,17</sup> ·

S. Nuwanthika Wijesinghe<sup>2,3</sup> · Jake Winiski<sup>19</sup> · Jiye Yan<sup>7</sup> · Erandi Yasanthika<sup>2,3</sup> · Marc Stadler<sup>12</sup>

Received: 5 April 2019 / Accepted: 20 May 2019 / Published online: 3 July 2019

© The Author(s) 2019

## Abstract

Fungi are an understudied, biotechnologically valuable group of organisms. Due to the immense range of habitats that fungi inhabit, and the consequent need to compete against a diverse array of other fungi, bacteria, and animals, fungi have developed numerous survival mechanisms. The unique attributes of fungi thus herald great promise for their application in biotechnology and industry. Moreover, fungi can be grown with relative ease, making production at scale viable. The search for fungal biodiversity, and the construction of a living fungi collection, both have incredible economic potential in locating organisms with novel industrial uses that will lead to novel products. This manuscript reviews fifty ways in which fungi can potentially be utilized as biotechnology. We provide notes and examples for each potential exploitation and give examples from our own work and the work of other notable researchers. We also provide a flow chart that can be used to convince funding bodies of the importance of fungi for biotechnological research and as potential products. Fungi have provided the world with penicillin, lovastatin, and other globally significant medicines, and they remain an untapped resource with enormous industrial potential.

**Keywords** Biocontrol · Biodiversity · Biotechnology · Food · Fungi · Mushrooms

## Table of contents

### From basic to applied research, prototypes and products

contribution by Birthe Sandargo, Marc Stadler

<figure><img src="image_2.png" /></figure>

Jianchu Xu
j.c.xu@cgiar.org

Extended author information available on the last page of the article

## Strategies against human disease

1. Antibacterial antibiotics
contribution by Clara Chepkirui, Benjarong Thongbai, Marc Stadler,

2. Antimycotics
contribution by Benjarong Thongbai, Marc Stadler

3. Biofilm inhibitors
contribution by Benjarong Thongbai, Marc Stadler
4. Anti-cancer agents
   contribution by Chayanard Phukhamsakda, Marc Stadler

5. Anti-diabetes
   contribution by Achala R. Rathnayaka, Marc Stadler

6. Improving nerve functioning
   contribution by Benjarong Thongbai, Marc Stadler

7. Fungi in Traditional Chinese Medicine
   contribution by Thatsanee Luangharn, Marc Stadler

8. Cardiovascular disease control by fungi
   contribution by Anuruddha Karunarathna, Marc Stadler

9. Antiviral agents
   contribution by Allan Patrick G. Macabeo, Marc Stadler

10. Immunosuppressive and immunomodulatory agents
    from fungi
    contribution by Clara Chepkirui, Marc Stadler

## Strategies against plant disease

11. Biocontrol of plant disease using endophytes
   contribution by Nimali I. de Silva, Siraprapa Brooks

12. Biocontrol of insects using fungi
   contribution by Allen Grace T. Niego

13. Biocontrol of nematodes and fungal nematizides
   contribution by Diana S. Marasinghe, Clara Chepkirui

14. Biocontrol of weeds and herbicides from fungi
   contribution by Pranami D. Abeywickrama, Jiye Yan

15. Fungal antagonists used in post-harvest disease control
   contribution by Binu C. Samarakoon

16. Bio control of rusts and smuts by antagonistic fungi
   contribution by Rashika S. Brahmanage

## Enhancing crops and forestry

17. Biofertilizers
   contribution by Mingkwan Doilom

18. Arbuscular mycorrhizae as biofertilizers
   contribution by Amornrat Chaiyasen, Saisamorn Lumyong

19. Application of ectomycorrhizal fungi in forestry
   contribution by Jaturong Kumla, Saisamorn Lumyong

20. Use of orchid mycorrhizae and endophytes in biotechnology
   contribution by Nimali I. de Silva, Sureeporn Nontachaiyapoom

21. Growth promoting hormones from fungi
   contribution by Saisamorn Lumyong

22. Mitigating abiotic stress in plants: the endophyte method

contribution by Karaba N. Nataraja, Uma Shaanker Ramanan

## Food and beverages from fungi

23. Growing mushrooms in compost
   contribution by Naritsada Thongklang

24. Growing mushrooms in bags
   contribution by Samantha Karunarathna

25. Growing mushrooms in the field
   contribution by Peter E. Mortimer, Samantha C. Karunarathna

26. Modern mushroom production: an automated factory process
   contribution by Jianchu Xu

27. New edible mushrooms
   contribution by Samantha Karunarathna

28. *Agaricus subrufescens*
   contribution by Naritsada Thongklang

29. Using fungi to enhance food value
   contribution by Danushka S. Tennakoon

30. Food colouring from filamentous fungi
   contribution by Wasan Sriprom and Saisamorn Lumyong

31. Food flavouring
   contribution by S. Nuwanthika Wijesinghe

32. What is mushroom stock? Products, process and flavours contribution by Deping Wei

33. Fungi in making tea
   contribution by Ningguo Liu, Jack JK Lui

34. Wine, beer and spirits
   contribution by Sinang Hongsanan

35. Functional foods and nutraceuticals
   contribution by Boontiya Chuankid

36. Harvesting the untapped probiotic potential of fungi
   contribution by Eleni Gentekaki, Achala R. Rathnayaka

## Saving the planet

37. Agricultural waste disposal
   contribution by Putarak Chomnunti, Craig Faulds

38. Mycoremediation: Fungi to the rescue
   contribution by Dulanjalee Harishchandra, Jiye Yan

39. Mycofumigation using *Muscodor*
   contribution by Nakarin Suwannarach, Saisamorn Lumyong

40. Biomass to biofuel: unmasking the potential of lesser-known fungi
   contribution by Venkat Gopalan, T.S. Suryanarayanan

41. Packed-bed bioreactor for mycomaterial production
   contribution by Peter Mueller, Dan Meeks, Meghan O'Brien, Jake Winiski
42. Fungal degradation of plastics: A hidden treasure for green environment
contribution by Sehroon Khan, Sadia Nadir

43. Polycyclic aromatic hydrocarbon degradation by basidiomycetes
contribution by Allen Grace T. Niego, Resurreccion B. Sadaba

44. Can fungi help modify the sustainable soil enhancer biochar?
contribution by Thitipone Suwunwong, Craig Faulds

## Commodities

45. Fungi and cosmetics
contribution by Erandi Yasanthika

46. Agarwood
contribution by S. Nuwanthika Wijesinghe

47. Fungal enzymes
contribution by Pattana Kakumyan

48. Preservatives
contribution by Benjarong Thongbai

49. Organic acids
contribution by Janith V.S. Aluthmuhandiram

50. Textile dyes
contribution by Ruvishika S. Jayawardena

## The future

Functional genomics and the search for novel anti-infectives

contribution by K.W. Thilini Chethana, Jiye Yan and Birthe Sandargo

## From basic to applied research, prototypes and products

Fungi have both good and bad facets (Pointing and Hyde 2001). They are essential for nutrient cycling because of their ability to degrade cellulose and lignin (Pointing et al. 2001). On the other hand, they cause serious human, animal and plant diseases and have numerous negative aspects on human life (Hyde et al. 2018a). Fungi are, however, also relatively understudied, but are an essential, fascinating and biotechnologically useful group of organisms with an incredible biotechnological potential for industrial exploitation. In this paper, we detail 50 ways in which we can potentially exploit fungi. We provide notes and examples for all potential exploitations and give examples from our own work and the work of others. We also provide a flow chart that can be used to convince funding bodies just how important fungi are and their potential for biotechnological research and potential products.

While several of our chapters are dealing with marketed products that even include blockbuster pharmaceuticals, such as the beta-lactam antibiotics, the statins and cyclosporine, others are dedicated to newly upcoming areas that still remain to be explored. Other chapters treat relatively small market segments that may expand in the future. For example, the consumers around the world now increasingly prefer natural compounds over synthetic chemicals and even in the industrial sectors that produce commodity chemicals, there is now an increased interest in development of sustainable biotechnological processes, in order to obtain new natural products that can eventually replace traditional synthetics. As compared to other biological sources, in particular plants, fungi have the great advantage that they can be grown in large bioreactors at an industrial scale, and suitable processes for their cost-efficient fermentation have been available for many decades, e.g. for production of certain organic acids, enzymes and antibiotics. As exemplified by the recent studies of the Thai mycobiota, modern polyphasic taxonomic approaches are constantly revealing a plethora of new and undescribed species even in the fairly well-known genera of fungi like *Agaricus* (Hyde et al. 2018b). Even the majority of the known species in the fungal kingdom are virtually untapped with regard to potential applications, also because they were never cultured and studied for their growth characteristics and physiology. New methods and protocols have to be developed for this purpose, and this implies that substantial basic research must be carried out before the exploitation of the novel organisms can be envisaged.

Although fungi have so many potential uses, research on their potential applications is in general poorly funded and much of the research that is being carried out in academia is fundamental, even in areas that belong to the fields of biotechnology and applied mycology. For example, screening fungi for production of antibiotics by antagonistic culture testing has often been reported, but is unlikely to lead to industrial projects. Often, it will take over a decade even to bring a given project based on a novel fungal metabolite into the preclinics, and even this is only possible by joint, interdisciplinary efforts of biologists, biotechnologists, pharmacists and chemists. Moreover, the Big Pharma industry has recently downsized their capacities for in-house research, meaning that the academic sector (sometimes supported by smaller companies or organisations like the Bill and Melinda Gates Foundation and the Wellcome Trust) has become more and more involved in the preclinical evaluation of new compounds.

Investing in basic research may seem, at first sight, a costly affair. However, there are numerous examples of the
past demonstrating why investing in basic research pays off in the long run, and even more reasons, why it is today more important than ever to renew an interest in basic research on fungi. But how to convince funders, in particular from the private sector, to invest into researchers doing basic research on fungi?

There are, no doubt, areas of research, which are of utmost importance to the entire world, yet are considered valueless to the pharmaceutical industry. One of these is the search for novel anti-infectives, as the world is running out of antibiotics (Hesterkamp 2017; WHO report 2017). It has long been seen as a tedious process to obtain novel antibiotics from living organisms.

However, the focus in the past has been on the same bacterial and fungal genera, such as *Streptomyces* in the Actinobacteria and common soil moulds like *Aspergillus* and *Penicillium* in the filamentous fungi (Karwehl and Stadler 2017). Since almost no novel carbon skeletons have been discovered from these common soil microbes in the past 20 years, it makes much more sense to study the numerous species that are constantly being discovered and shown to belong to new phylogenetic groups.

In our review, we present fungi, in particular Basidiomycota, as a still underexplored, highly promising source of anti-infectives, immunosuppressants, and other pharmaceuticals (see Badalyan et al. 2019; Sandargo et al. 2019a) that is nowhere near dried up. We give examples on recent developments of turning fungal natural products into commercial drugs and give an overview of the current state of applied research in this field.

In the past, fungal natural products have also led to some blockbusters and various developmental candidate compounds for the agrochemical industry (Bills and Gloer 2016). However, the uncontrolled usage of such fungal pesticides has led to the development of more and more resistances against these agrochemicals (Lucas et al. 2015). A more controlled approach of crop protection is therefore advisable. More basic research is needed to understand natural processes, and thereby allow for the search of natural control agents. In the entries dealing with “Strategies against plant disease”, we show the great potential of fungi as biocontrol agents. We give examples of how fungal biocontrol agents can help save the Agro sector tremendous amounts of money, if companies are given the opportunity to produce cost efficient biocontrol agents. In a likewise manner, the part on “Enhancing crops and forestry” deals with the current research on ectomycorrhiza and their potential application as natural biofertilizers.

With the new trend to a more sustainable, health-oriented living, and constant reports of hazardous chemicals found in food and cosmetics, the demand for more ecological, more “natural” alternatives is high. This is again, where fungi can step in. In the entries on “Food and

beverages from fungi” and “Commodities”, we present examples of how basic research on fungi has made its way into the food and beverage, but also the textile and flavour industry. Finally, in the part on “Saving the planet” we illustrate the great potential of fungi towards a more sustainable living and how fungi can assist to cope with some potential future challenges that are threatening human civilisation. A diagram illustrating all potential beneficial uses of fungi that are treated herein is given in Fig 1.

## Strategies against human disease

The scientific community recently celebrated the 90th anniversary of Sir Alexander Fleming's discovery of penicillin, which marked the starting point of the era of antibiotic chemotherapy. As outlined by Karwehl and Stadler (2017), among the numerous antibiotics that were discovered over the next 50 years, relatively few compound classes were derived from fungi. The latter include the cephalosporins (Newton and Abraham 1955), which belong to the same class as the penicillins, i.e. the beta glucan antibiotics, as well as fusidic acid (Godtfredsen et al. 1962) and pleuromutilin (Novak and Shlaes 2010; Sandargo et al. 2019a). Their chemical structures (1–4)<fnref n="1" /> are depicted in Fig. 2. Most other commercial antibiotics are actually derived from *Streptomyces* species and other actinobacteria, or even from other prokaryotes. For details of the history of research on antibiotics, we refer to the review by Mohr et al. (2017), as this does not fall within the scope of the current paper. As we cannot cover the entire field in this paper, we will give a brief overview on antibacterials, antimycotics and biofilm inhibitors and illustrate their usages with some examples of marketed drugs as well as other compounds that have recently been discovered.

### 1. Antibacterial antibiotics

The term “antibiotics” is used in the literature with different definitions. The industry mainly use it for antibacterial agents, but the definition that we prefer here, which was adapted from the original one coined by Waksman (1947), i.e., an antibiotic is “*a chemical substance, produced by micro-organisms (including fungi), which has the capacity to inhibit the growth of and even to destroy bacteria and other micro-organisms*”. The natural functions of antibiotics can easily be explained, resulting from the high competition between fungi, bacteria and other organisms in

<footnote n="1">¹ All chemical structures of fungal secondary metabolites presented in this paper have been numered consecutively in **bold** typeset in both, the figures and the corresponding text.</footnote>
<figure><img src="image_3.png" /><figcaption>**Fig. 1** Diagram showing the potential use of fungi in biotechnology. The cycle starts with basic biodiversity research, which in turn leads to cultures placed in the central culture collection. The cultures are</figcaption></figure>

substrates such as soil, dung and plant debris. If a given
organism has acquired the ability to produce a certain
secondary metabolite by which it can kill the competing
organisms that dwell in the same habitat, it is considered to
possess a selective advantage that ultimately increases its
fitness (Shearer 1995). Therefore it should come as no
surprise that one large experimental study concluded that
the majority of filamentous fungi are able to produce

then used for applied research, which in turn leads to products in the
form of the items discussed in the entries of this paper

antibiotic compounds (Bills et al. 2009). Bills and Gloer
(2016) summarized numerous important facts concerning
the current state of the art in research on fungal secondary
metabolites and concentrated heavily on the biochemical
and genetic background of their biosynthesis.

We are currently living in the “post-antibiotic” era,
where both, the numbers and percentages of multi-resistant
bacterial and fungal pathogens against the established
<figure><img src="image_4.png" /><figcaption>**Fig. 2** Chemical structures of fungal metabolites that were developed to antibacterial drugs</figcaption></figure>

antibiotics are drastically increasing, while the number of new therapeutic agents and developmental candidates has decreased (Cooper and Shlaes 2011). The reasons for this development are manifold, but the phenomenon is primarily due to the fact that the majority of pharmaceutical companies have lost interest in Research and Development on natural products and/or given up their activities in the anti-infectives sector. Experts around the world are now giving warnings about the serious consequences that the lack of antibiotics—in particular against the multi-resistant Gram negative human pathogenic bacteria—can have (Friedman et al. 2016). After two decades of neglect, efforts of both the private and the academic sector on the discovery of new antibiotics have substantially increased.

The pipeline for antibacterial antibiotics (Hesterkamp 2017) shows that there are still some compounds under development, but the majority of those have been optimised from old compounds with known modes of action, e. g. by chemical modifications. Therefore, it is likely that the resistant pathogens will easily find a way to cope with the new products, once they have reached the market. The aforementioned mutilins, which are derived from fermentation of the basidiomycete *Clitopilus passeckerianus* and subsequent semisynthesis, therefore represent the “newest” compound class that has been registered as an antibacterial drug. A derivative, retapamulin (5), was launched for use as a topical antibiotic against skin infections, and several further derivatives are undergoing clinical trials as systemic antibiotics. In general, basidiomycete cultures are much more difficult to handle with respect to large scale production of secondary metabolites, since they grow rather slowly and often have low yields. For the production of pleuromutilin, however, Bailey et al. (2016) managed to increase the yields substantially after the transfer of the biosynthetic genes into a fast growing heterologous

*Aspergillus* host, which can more easily be handled in the production process. This accomplishment can give rise to some hope that in the future, more of the hitherto neglected, unique biologically active metabolites of basidiomycetes can be made accessible to preclinical development.

## 2. Antimycotics and fungicides

Whereas multi-resistant bacterial pathogens are very high on the agenda of both the press and funding agencies, relatively little attention is presently being paid to the fact that the number of resistant pathogenic fungi is also on the rise. This topic was treated by Hyde et al. (2018a), we refer to it for the most important ant threatening human pathogenic fungal organisms. In fact, there are only a handful of efficient compound classes on the market that are used in antimycotic chemotherapy, including griseofulvin (6), which was already discovered by Grove et al. (1952; Fig. 3). The newest class of antimycotics that were launched to the market are the echinocandins (e.g., pneumocandin B₀ (7) (Denning 2002). The biosynthesis of these highly complex lipopeptides relies on PKS-NRPS hybrid gene clusters (Chen et al. 2013). They are being produced biotechnologically by large scale fermentation using different fungi that are not phylogenetically related and subsequent semisynthesis. The knowledge about the molecular mechanisms of their biosynthesis may in the future lead to the concise manipulation of the production process that can be directed towards new natural derivatives. Interestingly, a comparative genomics study by Yue et al. (2015) has revealed rather high homologies among the biosynthesis gene clusters of the producer organisms that belong to three different classes of Ascomycota, namely Dothideomycetes, Eurotiomycetes, and Leotiomycetes. Possibly, this has
<figure><img src="image_5.png" /><figcaption>**Fig. 3** Chemical structures of fungal metabolites with antifungal activity against human pathogens</figcaption></figure>

been due to horizontal gene transfer during the evolutionary history of these organisms.

Recent efforts aimed at the discovery of novel antifungal agents have resulted in a number of developmental projects, such as enfumafungin (8) from *Hormonema* spp. (Peláez et al. 2000). This compound class may soon yield the first pharmaceutical drug for use in humans that originated from a fungal endophyte, over 15 years after their first discovery. Even the biosynthesis genes encoding for these unique triterpenoids (Fig. 3) has only recently been identified (Kuhnert et al. 2018).

The search for novel antimycotics and fungicides has also resulted in the rediscovery of “old” compounds that may become more interesting in the future because they have originally been found in a screening for agrochemical fungicides and were never evaluated for their effects on human fungal pathogens or their mode of action. While the strobilurins, which are a very commercially successful antifungal agents in agriculture (Sauter et al. 1999), have been found inefficient or too toxic for application in humans, many other metabolites with pronounced antifungal effects were apparently never tested on their efficacy against human pathogens. A recent example for such rediscoveries is favolon (9), which is actually a co-metabolite of strobilurins produced by the invasive basidiomycete *Favolaschia calocera* (Chepkirui et al. 2016) and was originally isolated by Anke et al. (1995). Like the sporothriolodes (10) from the xylarialean fungus *Hypoxylon monticulosum* (Surup et al. 2014; Fig. 3; now classified in the new genus *Hypomontagnella* as *H. monticulosa*; cf.

Lambert et al. 2019), this metabolite shows very strong antifungal effects that are not accompanied by prominent cytotoxicity.

### 3. Biofilm inhibitors

Scientists are exploring different avenues to combat infectious diseases caused by both bacterial and fungal pathogens, for which the inhibition of biofilm formation is one of the most promising leads. Abraham and Estrela (2016) reported that fungal metabolites are becoming increasingly explored for their potential to inhibit the formation of biofilms, e.g. by interfering with quorum sensing, and some compounds have already been discovered that can even destroy pre-formed biofilms. A recent example is coprinuslactone (11) (de Carvalho et al. 2016; Fig 3), a small molecule derived from the edible mushroom *Coprinus comatus*, which acts against *Pseudomonas aeruginosa* biofilms. Other examples include roussoellenic acid (12) from a *Roussella* sp. (Phukhamsakda et al. 2018), which is active against biofilm formation in *Staphylococcus aureus*, as well as microporenic acid A (13) from a Kenyan basidiomycete (Chepkirui et al. 2018; Fig. 4), which can not only inhibit biofilm formation in both *Staphylococcus aureus* and the human pathogenic yeast, *Candida albicans*, but even destroys pre-formed biofilm in *C. albicans* at rather low concentrations. These compounds do not have prominent antimicrobial activities and therefore their application is unlikely to raise resistance. The biofilm inhibitors are very promising candidates for use in
<figure><img src="image_6.png" /><figcaption>**Fig. 4** Chemical structures of fungal metabolites with biofilm inhibition activities</figcaption></figure>

combination therapy with antibiotics. In several studies, biofilm inhibitors were shown to enhance the activity of the antibiotics by increasing their ability to penetrate the biofilms.

These examples illustrate that fungi are under-explored with respect to novel antibiotics and other therapeutic agents, and that it is certainly worthwhile to expend more effort in this area of research with an emphasis on hitherto neglected species from regions and habitats that have not yet been studied systematically. Fungi have much to offer in terms of novel chemistry: due to the advent of revolutionary techniques in genomics, transcriptomics, bioinformatics, analytical chemistry and biotechnological process development, we can now explore the chemical diversity of the mycobiota much more concisely than ever before. Evidence is also accumulating that novel phylogenetic lineages or hitherto neglected taxonomic and ecological groups of fungi can now much more easily be recognized and subjected to the exploitation of their secondary metabolome. However, more public funding is needed to assure that the substantial know-how that has been acquired over many decades does not become forgotten, and that the next generation of researchers will also still be able to work on novel, hitherto unexplored fungal groups, rather than only on model organisms.

## 4. Anti-cancer agents

Cancer is the second leading cause of mortality after cardiovascular disease, with an estimated 9.6 million cancer-related deaths in 2018 (GBD 2015). Cancer is a multifactorial disease characterized by the loss of growth factors that control the proliferation and division of cells. These abnormal malignant cells can evade the tumour suppressor factors of the human immune system, then develop to tumours and destroy adjacent tissues (Saeidnia and Abdollahi 2014). There are several treatments for cancer, administered according to developmental state of the disease. Chemotherapy, radiation therapy, surgery and

immunotherapy are all important elements of cancer treatment. However, while many cytotoxic agents are known to Science (which could in principle serve as chemotherapeutic agents), only few of them specifically target tumour cells and are less toxic to regular, healthy human tissue (Petrelli et al. 2012; Cai et al. 2013; Zugazagoitia et al. 2016). Targeted therapy, usually the conjugated element for cancer treatments, delivers drugs to genes or proteins that are specific to cancer cells or the environmental tissues that promotes the growth of cancer (Padma 2015). Fungi are an importance source for natural product discovery, albeit most anticancer drugs are retrieved from plants and bacteria. In this entry, we describe several promising natural products derived from fungi and highlight some of the chief compounds that are currently in the clinical and preclinical developmental stage (Fig. 5).

Irofulven (14) is a semi-synthetic derivative of illudin S (15), a natural toxin isolated from *Omphalotus illudens* (Jack O'Lantern mushroom; cf. Chin et al. 2006; Movasaghi et al. 2006). Irofulven interferes with DNA replication-complexes and cell division in DNA synthesis. The abnormal cells in S-phase lead to apoptotic cell death (Walser and Heinstein 1973; Jaspers et al. 2002). The antitumour activities of irofulven have been evaluated in phase I and II clinical trials with promising results against a variety of cancers, including those in the brain and central nervous system, breast, blood, colon, sarcoma, prostate, lungs, ovarian and pancreas (Alexandre et al. 2004; Miyamoto et al. 2018; Topka et al. 2018). Sandargo et al. (2019a) have recently described the state of the art, including some exciting new illudin conjugates that show superior in vitro activities than irufulven and are presently under early preclinical development.

Aphidicolin (16) is a tetracyclic diterpene with antiviral and antimitotic properties. The compound was originally isolated from "Cephalosporium aphidicola" (currently valid name: *Akanthomyces muscarius*) and later also reported from *Nigrospora sphaerica* (Bucknall et al. 1973; Starratta and Loschiavo 1974). Aphidicolin competes for the specific binding site on DNA polymerase α, δ, and ε enzymes. Its mechanism of action and efficacy have been intensively tested in clinical trials (Crosetto et al. 2013; Ayob et al. 2017), but so far it has not become a marketed drug.

Other anticancer lead compounds derived from fungi include leptosins F (17) and C (18) isolated from *Lep-toshaeria* sp., which showed antitumor activity in mouse embryos (Yanagihara et al. 2005; Pejin et al. 2013); β-glucans, the polysaccharides that are naturally found on the cell walls of fungi (Chan et al. 2009; Bashir and Choi 2017); as well as palmarumycin (Powis et al. 2006) and spiropreussione A (Chen et al. 2009). The latter
<figure><img src="image_7.png" /><figcaption>**Fig. 5** Chemical structures of promising natural products derived from fungi with anticancer activity</figcaption></figure>

compounds, however, have only demonstrated these activities in vitro, and it is not clear whether they will eventually reach the late exploratory stage of preclinical development.

## 5. Anti-diabetes

Diabetes mellitus, also known simply as diabetes, is a chronic metabolic disorder (De Silva et al. 2012). People who suffer from diabetes cannot produce or effectively use insulin in the body. Due to this insulin imbalance, they have high amounts of glucose in their blood. There are two common types of diabetes, i.e. type 1 diabetes (insulin dependent diabetes mellitus) and type 2 diabetes (noninsulin-dependent diabetes mellitus).

Patients with type 1 diabetes cannot produce insulin, due to the lack of functions of the insulin-secreting beta cells in the pancreas (Meier et al. 2005). They must take insulin continuously every day to stay alive. Type 1 diabetes mostly affects children and adolescent patients, and it represents 5–10% of total diabetes cases worldwide. Patients with type 2 diabetes cannot produce sufficient insulin or cannot effectively metabolize it. This form of the disorder commonly affects elderly people and accounts for 90–95% of all diabetes cases (Hameed et al. 2015). People worldwide suffer from diabetes mellitus and 7% of the world's adult population is affected by the disease (Philippe and Raccah 2009). In 2017, the largest number of

diabetic patients, around 114 million, was recorded in China. Roughly 73 million diabetic patients were recorded in India, and 30 million were recorded in the United States. (https://www.statista.com/statistics/281082/countries-with-highest-number-of-diabetics/).

There are many negative consequences for patients if diabetes remains untreated, such as blindness, kidney failure, depression, cardiovascular diseases, cancer and even death (Gerstein et al. 2011; Hansen et al. 2012; Huang et al. 2018). Retinopathy (damage of the retina, leading to blindness) and neuropathy (damage of the nervous system) are some of the most severe complications that have been attributed to diabetes (De Silva et al. 2012; Sobngwi et al. 2012).

Many Basidiomycota, such as *Agaricus bisporus*, *Cyclocybe aegerita*, *C. cylindracea* and *Tremella fuciformis* are used as medicine for the treatment or prophylaxis of type 2 diabetes. These mushrooms help patients avoid high levels of glucose because they contain the least amount of digestible carbohydrates in the diet (Poucheret et al. 2006). Bioactive metabolites, which are isolated from medical mushrooms and their cultured mycelia, act as biological antihyperglycemic agents in diabetes treatment (Table 1) (De Silva et al. 2012). Extracts of *Inocutis levis* (Hymenochaetaceae) have been reported to possess utility as a remedy for diabetes because they increase insulin resistance, insulin sensitivity and glucose uptake in tissues and hence help to control blood glucose levels (Ehsanifard
<table><caption>**Table 1** Anti-diabetic effects of some medical mushroom species</caption><thead><tr><th>Mushroom species</th><th>Common name</th><th>Bioactive compounds</th><th>Experimental setup and observed effects</th><th>References</th></tr></thead><tbody><tr><td>Agaricus bisporus</td><td>White button mushroom</td><td>Dry fruiting body extract</td><td>Streptozotocin-injected diabetic Sprague Dawley rats (reduced blood glucose level)</td><td>Jeong et al. (2010)<br>Yamac et al. (2010)</td></tr><tr><td>Agaricus campestris</td><td>Field mushroom</td><td>Aqueous fruiting body extract</td><td></td><td>Gray and Flatt (1998)</td></tr><tr><td>Agaricus subrufescens</td><td>Almond mushroom</td><td>β-Glucans from dried fruiting bodies</td><td></td><td>Kim et al. (2005)</td></tr><tr><td>Inonotus obliquus</td><td>Chaga mushroom</td><td>Ethyl acetate fraction from dried fruiting bodies</td><td>Alloxan-induced diabetic mice (reduced blood glucose level)</td><td>Lu et al. (2010)</td></tr><tr><td>Ophiocordyceps sinensis (“Cordyceps” sinensis)</td><td>Caterpillar fungus</td><td>Polysaccharide from Ophiocordyceps (Cordyceps) mycelia</td><td>Streptozotocin- and alloxan-induced diabetic mice (reduced blood glucose level/increased the blood insulin level)</td><td>Li et al. (2003)<br>Li et al. (2006)</td></tr><tr><td>Hericium erinaceus</td><td>Lion's mane mushroom</td><td>Methanol extract of the dried fruiting body</td><td>Streptozotocin-induced Wistar rats (reduced blood glucose level)</td><td>Wang et al. (2005b)</td></tr><tr><td>Tremella mesenterica</td><td>Yellow brain mushroom</td><td>Fruiting body extract</td><td>Streptozotocin-induced Wistar rats (increased insulin secretion and glucose metabolism)</td><td>Lo et al. (2006)</td></tr></tbody></table>

et al. 2017). The fruiting bodies of *Antrodia cinnamomea* can be used to produce healthy foods and drugs that have anti-diabetes properties (Huang et al. 2018). *Grifola frondosa* has been used as medicine for type 2 diabetes, and its extracts can effect both hyperglycemia (when a high amount of glucose circulates in the blood) and hyperinsulinemia (when a high level of insulin circulates in the blood) (Poucheret et al. 2006).

Fungal products are sold as remedies for diabetes. *Ophiocordyceps sinensis* capsules, SX-Fraction, Reishi-Max capsules and *Tremella* are some of the examples of anti-diabetic products made with medicinal mushrooms (Li et al. 2004; De Silva et al. 2012) and is claimed to decrease fasting blood glucose levels in type 2 diabetes. This medicine can also be used to reduce blood pressure and body weight. SX-Fraction is considered a major alternative for enhancing insulin sensitivity (Preuss et al. 2007). *Tremella* is a medicinal product used in Chinese medicine; produced from *Tremella fuciformis* (silver ear mushroom/ white jelly leaf mushroom), it is mainly used for reducing blood glucose and cholesterol levels (Li et al. 2004).

Future investigation is needed to clarify the long-term effects of taking medicinal mushroom products with other drugs. It is necessary to justify the use of medicinal mushroom products as anti-diabetes (De Silva et al. 2012).

## 6. Improving nerve functioning

Human neurodegenerative diseases, such as Alzheimer's, Huntington's, and Parkinson's disease, are disrupting neuronal populations in adults worldwide. The discovery and development of neuroactive compounds from medicinal mushrooms with the potential to improve nerve functioning has been extensively studied. Mushrooms, such as *Antrodia camphorata*, *Ganoderma* spp., *Hericium erinaceus*, *Lignosus rhinocerotis* and *Pleurotus giganteus*, have a long history of use in enhancing the peripheral nervous system. Nerve growth factors are important for the survival, maintenance, and regeneration of specific neuronal populations in the adult brain. It has been demonstrated that neurodegenerative diseases mostly occur because of the disappearance of nerve growth factors. Therefore, scientists have been attempting for over 20 years to discover fungi-derived neuroactive components which are able to cross the blood-brain barrier and induce the production of nerve growth factors.

Most of the potential neuroactive compounds, which may aid in the prevention or therapy of neurodegenerative diseases, have been discovered in *Hericium erinaceus*. This medicinal mushroom is known to produce two unique terpenoid classes, namely hericenones and erinacines, from its fruiting body and mycelia, which can stimulate synthesis of the nerve growth factor via the TrkA/Erk1/2 pathway. Not

<figure><img src="image_8.png" /></figure>
only does *H. erinaceus* induce nerve growth factors or nerve regeneration, but it has also been shown to improve digestive functioning and effect relief from gastritis while providing immune-support, such as anti-inflammatory and anti-oxidant activities. Several studies have demonstrated that hericenones such as hericenone A (19) and erinacines (erinacine C (20) (Fig. 6) induce the synthesis of nerve growth factors in vivo and in vitro (Thongbai et al. 2015). Wittstein et al. (2016) discovered corallocins A–C, a nerve growth and brain-derived neurotrophic factor inducing metabolites, from the related *Hericium coralloides* in cell based bioassays, while Rupcic et al. (2018) discovered two new erinacine derivatives from mycelial cultures of *H. erinaceus* and *H. flagellum*. Additional studies have revealed neuroactive activities in extracts from medicinal mushrooms that induce nerve growth factors, but the active principles remain to be identified. For instance, the aqueous sclerotium extract from the Malaysian medicinal mushroom, *Lignosus rhinocerotis*, contained neuroactive compounds that have been demonstrated to stimulate neurite outgrowth in vitro (Eik et al. 2012). Similarly, aqueous extracts of fruiting bodies of species referred to as “Ganoderma lucidum” (questionable identification because the report was from Asia where this species does not actually occur) and *G. neo-japonicum* were also effective at stimulating neurite outgrowth (Seow et al. 2013). *Pleurotus giganteus* was found to contain a high concentration of uridine, which has also shown nutritional outgrowth stimulatory effects (Phan et al. 2012). These results may give rise to a more systematic study of the phenomenon in the future.

The family Hericicaceae comprises other genera and species, such as *Laxitextum incrustatum* Mudalungu et al. (2016) and *Dentipellis fragilis* (Mitschke 2017), which were proven to contain compounds of the erinacine type; these cyathane terpenoids may constitute family-specific markers. On the other hand, Bai et al. (2015) have reported

cyathane diterpenoids as nerve growth factor enhancers from cultures of a *Cyathus* species, and even the cyathanes from the fruit bodies of the mycorrhizal basidiomycetes of the genus *Sarcodon* seem to have similar effects (Cao et al. 2018). It should therefore be worthwhile to study genera that are known to produce cyathanes, as well as representatives of other mushroom genera related to *Hericium*, including *Amylosporus* and *Wrightoporia*, for such phenomena. This examination may lead to the discovery of additional novel neurotropic compounds. Reliable screening systems as well as genetic models of human neurodegenerative diseases are now available for in vivo cell biological analysis of disease progression and intervention.

While the mode of action of the hericenones, corallocins and erinacines remains to be clarified, a very important drug that constitutes a mimetic of a fungal metabolite should be mentioned in this context. Fingolimod (21) is a product of total synthesis that has been discovered during the course of a mimetic synthesis program that used as template myriocin (22), a compound produced by the insect associated ascomycete *Isaria sinclairii* (Strader et al. 2011) (Fig. 6). Fingolimod (sold under the brand Gilenya), is a potent immunosuppressant that was approved in 2010 by the U.S. Federal Drug Association as a new treatment for multiple sclerosis. Fingolimod is phosphorylated in vivo by sphingosine kinase 2, and the resulting metabolite binds to the extracellular G protein-coupled receptors, sphingosine 1-phosphates. This prevents the release of lymphocytes from lymphoid tissue and therefore can suppress the immune system. Aside from its proven effects against the symptoms of multiple sclerosis, the compound can also potentially be used in the therapy of cancer and during organ transplants. Likewise, additional potential usages for the erinacines and hericenones may become conceivable once the biochemical and molecular mechanisms by which they exert their activities in biological systems have been elucidated.

<figure><img src="image_9.png" /><figcaption>**Fig. 6** Chemical structures of neurotrophic compounds from *Hericium*, as well as myriocin and its synthetic analogue fingolimod</figcaption></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>
## 7. Fungi in Traditional Chinese Medicine

Traditional Chinese Medicine (TCM) has been used for thousands of years, during which time traditional knowledge originating in ancient China has been gradually developed further and distributed to Japan and other Asian countries. TCM relies heavily on the use of natural remedies including plants, animals, and even minerals to cure various diseases. However, in spite of the fact that the term “herbal medicine” is widely used to describe TCM and other ethnobotanical approaches for disease therapy, several mushrooms are commonly used as important ingredients. Furthermore, since fungal taxonomy has yet to be globally harmonized, there is considerable confusion concerning the identity of these important traditional “herbal” remedies. A compilation of the most important TCM mushrooms is therefore provided in Table 2, and we will henceforth refer to the species according to their scientific names as given in this table, regardless of whether they have been or are still being referred to by incorrect or ambiguous names. For instance, we will refer to

“Ganoderma lucidum” as *G. lingzhi*, regardless of whether the authors of the respective cited papers had already adopted the modern taxonomy.

TCM has evolved through a combination of theory and practical experience of Chinese healers over many centuries. The first recorded use of Chinese medicine was during the Shang Dynasty in the eleventh century BC (1100–1001 BC), where even *Ganoderma* was recorded in detail and for the first time. However, the first comprehensive description of Chinese herbal remedies and their medicinal value was published in “Shennong Bencaojing ( bencao ),” during the Han dynasty (206 BC-220 AD). Later, another very comprehensive account of Chinese herbology, known as the “Bencao Gangmu ( bencao )” or “Compendium of Materia Medica” (Sanodiya et al. 2009) was compiled by Li Shizhen (1518 ~ 1593) during the Ming Dynasty (1368–1644). In 1987, Chinese scientists compiled a list of 272 medicinal mushroom species (Ying et al. 1987). Later, this number increased to 799 species, 500 thereof with antitumor effects (Wu et al. 2013), and we estimate that about 850 species are presently being used as

<table><caption>**Table 2** The correct scientific terminology of some important medicinal mushrooms used in traditional Chinese medicine</caption><thead><tr><td>Current name</td><td>Important synonyms/misapplied names</td><td>Literature</td></tr></thead><tbody><tr><td>Agaricus subrufescens<br>(巴西蘑菇: Bā xī mó gū)</td><td>Agaricus blazei (ss. Heinemann) (s. auct.); A. rufotegulis (s. auct.); Agaricus brasiliensis</td><td>Kerrigan (2005)</td></tr><tr><td>Cordyceps militaris<br>(虫草花: Chóng cǎo huā)</td><td>Clavaria militaris L. (s. auct.); Torrubia militaris (s. auct.)</td><td>Shrestha et al. (2010), Sung et al. (2007), Baroni (2017)</td></tr><tr><td>Ganoderma lingzhi<br>(靈芝: Líng zhī)</td><td>Ganoderma lucidum (s. auct.); G. sichuanense (s. auct.)</td><td>Cao et al. (2012), Dai et al. (2017)</td></tr><tr><td>Ganoderma sinense<br>(紫芝: Zǐ zhī)</td><td>Ganoderma formosanum (s. auct.)</td><td>Wang et al. (2005a), Thawthong et al. (2017)</td></tr><tr><td>Grifola frondosa<br>(灰树花: Huī shù huā)</td><td>Boletus frondosus (s. auct.) Polyporus frondosus (s. auct.)</td><td>Corner (1989), Shen et al. (2017)</td></tr><tr><td>Hericium erinaceus<br>(猴头菇: Hóu tóu gū)</td><td>Hydnum erinaceus (s. auct.)</td><td>Zhou et al. (2011), Thongbai et al. (2015)</td></tr><tr><td>Lentinula edodes<br>(香蕈: xiāng xùn)</td><td>Lentinus edodes (s. auct.)</td><td>Molina et al. (1992), Hibbett and Vilgalys (1993), García-Mena et al. (2007), Vetchinkina et al. (2008)</td></tr><tr><td>Lycoperdon pyriforme<br>(梨形马勃: Lí xíng mǎ bó, or<br>马勃属: Mǎ bó shù)</td><td>Morganella pyriformis (s. auct.)</td><td>Krüger and Kreisel (2003), Larsson and Jeppson (2008)</td></tr><tr><td>Ophiocordyceps sinensis<br>(冬虫夏草: Dōng chóng xià<br>cǎo)</td><td>Cordyceps sinensis (s. auct.)</td><td>Shrestha et al. (2010), Zhang et al. (2012)</td></tr><tr><td>Trametes versicolor<br>(雲芝: Yún zhī)</td><td>Coriolus versicolor (s. auct.); Polyporus versicolor (s. auct.)</td><td>Tomšovský and Homolka (2004), Justo and Hibbett (2011), Wan (2013)</td></tr><tr><td>Wolfiporia cocos<br>(茯苓: Fú líng)</td><td>Poria cocos (s. auct.)</td><td>Kubo et al. (2006), Dai et al. (2009)</td></tr><tr><td>Xylaria nigripes<br>(黑柄炭角菌: Hēi bǐng tàn<br>jiǎo jūn)</td><td>Pseudoxylaria nigripes (s. auct.)</td><td>Rogers et al. (2005)</td></tr></tbody></table>

<figure><img src="image_13.png" /></figure>
<figure><img src="image_14.png" /><figcaption>**Fig. 7** Different dried *Ganoderma* products sold in a Traditional Chinese Medicine pharmacy market, Kunming Province, China</figcaption></figure>

remedies in China. TCM has adopted a more holistic philosophy when compared with Western medicine (Wang et al. 2017). Chinese medicinal mushrooms have been used since the beginning of human history as a “protector herb” in order to maintain and enhance good health.

Whereas the majority of the TCM ingredients are derived from plants, several macrofungi have been included in the formulas. Some macrofungi are frequently included in TCM medicine formulas and are also very commonly sold in the medicinal or local markets (Fig. 7). The most important species and species groups are compiled in Table 2. Notably, some, such as *Ganoderma lingzhi* and *Wolfiporia cocos*, are very expensive, while others such as *Ophiocordyceps sinensis* have become rare in nature (Sung et al. 2007; Zhang et al. 2012; Xiang et al. 2014; Hapuarachchi et al. 2018, 2019).

*Ganoderma lingzhi* (Lingzhi) contains both complex, high and low molecular weight natural bioactive compounds, which are mostly triterpenoids (Sandargo et al. 2019a). Bioactive compounds have shown in vitro activities against cancer cells, although none have been regarded as effective enough to enter clinical development as an ethical anticancer drug. Extracts of mycelial cultures and fruiting bodies of *Ophiocordyceps sinensis* and *Cordyceps militaris* primarily contain cordycepin, a derivative of nucleoside adenosine, with dimethylguanosine and iso-sinensetin, and showed moderate anticancer and antiproliferative effects in vitro (Wong et al. 2010). Several bioactive compounds derived from mushrooms used in TCM were identified by bioactivity-guided fractionation. On the other hand, the potential therapeutic value of these fungal TCM products for the treatment of various diseases (e.g. cancer, diabetes, cardiovascular and neurodegenerative diseases) is even supported by clinical studies (Table 3).

There has been intensive debate over the utility of TCM for modern healthcare. Many clinical scientists have doubted the utility of the classical TCM remedies because of their inaccurate descriptions, as well as the different philosophies regarding their therapeutic application. In

fact, therapy for diseases in the Western world is mainly based on the application of single substances that have strong and selective biological and pharmacological activities, and must undergo very thorough clinical trials before they can be applied as therapeutic drugs. TCM, by contrast, often uses mixtures of preparations made from several plants, fungi and other organisms, each of which may contain dozens if not hundreds of different compounds. Therefore TCM does not really fit into the modern healthcare system (Fu and Yu 2005). However, the example of artimisinin shows that compounds from TCM can be very useful as remedies for diseases that have an unmet medical need. This terpenoid was originally found in the asteraceous traditional Chinese medicine plant *Artemisia annua*; a semisynthetic derivative, artesunate, is now on the market as a last generation antimalaria drug (the inventor received the Nobel Prize for Medicine in 2015). This example shows that it is worthwhile to study the ingredients of traditional Chinese medicine plants using modern drug research methods in order to discover novel therapeutic agents. Several examples of beneficial compounds from mushrooms have found their way into exploratory drug research projects (Grothe et al. 2011).

In addition, the recent integration of TCM and modern medicine has begun to solve multiple worldwide health problems. Many databases were evaluated to advance scientific formulations, chemical analysis, potential approaches, and other health targets. The World Health Organization (WHO) has also released a standard series for developing traditional medicines across the world, including medicinal mushrooms (Tang et al. 2018). The discovery of biologically active compounds from medicinal mushrooms can impact the direction of future medical development, and also has broad market prospects in North America, Europe and other developed economies (Lee et al. 2012; Wang et al. 2017). This may also increase international exchange, leading to the unprecedented development of Chinese medicine in the western world (Zhu 2018).
<table><caption>**Table 3** Some important medicinal mushrooms used in traditional Chinese medicine</caption><thead><tr><th>Fungal species</th><th>Therapeutic use (diseases)</th><th>Instructions</th><th>References</th></tr></thead><tbody><tr><td>Agaricus<br>subrufescens</td><td>Anticancer, anti-inflammatory, anti-allergic, hepatoprotective, diabetic, arteriosclerosis and (treat kidney disease, bowel disease, asthma, diabetes and high blood sugar levels)</td><td>P, S, and T</td><td>Grothe et al. (2011), Kerrigan (2005), Jeong et al. (2010), Lee et al. (2012)</td></tr><tr><td>Cordyceps<br>militaris</td><td>Anticancer, antioxidant, antibacterial, antifungal, antiviral, antihypertensive, diabetic, anti-fibrotic, anti-angiogenesis, and hepatoprotective (treating kidney disease, lung fibrosis, high blood pressure, high blood sugar levels, and strengthen sexual function)</td><td>B, M, and S</td><td>Lee et al. (2012), Xiang et al. (2014)</td></tr><tr><td>Ganoderma<br>lingzhi</td><td>Immunostimmulatory, anticancer, anti-inflammatory, antiviral, antioxidant, antibacterial, antifungal, anti-hypotensive, anti-metastatic effect and diabetic (treat kidney, liver and lung diseases, asthma or bronchial, insomnia, neurasthenia, and strengthen sexual function)</td><td>B, P, S, SP, and T</td><td>Liu et al. (2002), Richter et al. (2015), Qiao et al. (2005), Cheng et al. (2010), Teng et al. (2011, 2012), Lee et al. (2012), Hapuarachchi et al. (2014)</td></tr><tr><td>Ganoderma<br>sinense</td><td>Immunostimmulatory, anticancer, antiviral, anti-inflammatory, antioxidant, antibacterial and antifungal (treating liver, chronic coughs, asthma and leukopenia)</td><td>B, P, S, SP, and T</td><td>Richter et al. (2015), Teng et al. (2011, 2012), Jiang et al. (2017)</td></tr><tr><td>Grifola frondosa</td><td>Anticancer, antiviral, anti-inflammatory, anti-hypotensive, hepatoprotective, and diabetic (treat liver, therapy of HIV, low blood pressure, and high blood sugar levels)</td><td>S and T</td><td>Chien et al. (2017), Ma et al. (2015a, b)</td></tr><tr><td>Hericium<br>erinaceus</td><td>Anticancer, neuroprotective hemostatic (used to treatment Alzheimer's and Parkinson's disease)</td><td>P, S, and T</td><td>Salmon (2012), Thongbai et al. (2015), Cheng et al. (2016), Yang et al. (2016), Zhang et al. (2017a)</td></tr><tr><td>Lentinula edodes</td><td>Anticancer, antiviral, antibacterial, anti-inflammatory, and antihypertensive (treatment of high blood pressure)</td><td>B, M, S, and T</td><td>Lee et al. (2012), Rincão et al. (2012), Lin et al. (2015)</td></tr><tr><td>Lycoperdon<br>pyriforme</td><td>Antibacterial, anti-inflammatory, antitumor, (treatment of pharyngitis; hemostasis)</td><td>M (young fruiting body), and SP</td><td>Akatin (2013), Qian et al. (2018)</td></tr><tr><td>Ophiocordyceps<br>sinensis</td><td>Anticancer, antiviral, anti-inflammatory, antioxidant, antibacterial, antifungal, anti-angiogenetic, anti-proliferative, hypoglycaemic, hypolipidaemic, renoprotective, hepatoprotective (treat heart, lung and kidney diseases, high blood sugar levels and strengthen sexual function)</td><td>M, P, S and T</td><td>Akihisa et al. (2009), Jeon et al. (2011), Lee et al. (2012), Xiang et al. (2014)</td></tr><tr><td>Trametes<br>versicolor</td><td>Antitumor, antiviral, anti-inflammatory, antimalarial activity, diabetic, and hepatitis (treat kidney disease, diabetes, malaria and HIV)</td><td>B, C and T</td><td>Fu and Yu (2005), Adekunle et al. (2016)</td></tr><tr><td>Wolfiporia cocos</td><td>Immunostimmulatory, antiviral, anti-inflammatory, antiasthmatic, hepatoprotective, anti-parasitic (treatment of liver and lung disease, asthma or bronchial)</td><td>C, P and T</td><td>Wang et al. (2013b), Wei et al. (2016), Zhu et al. (2018)</td></tr><tr><td>Xylaria nigripes</td><td>Anticancer, antiviral, anti-inflammatory, hypoglycaemic, hepatoprotective (treating kidney disease, gynecological diseases, and therapy of insomnia)</td><td>B, C and T</td><td>Fu and Yu (2005), Peng et al. (2015b)</td></tr></tbody></table>

B boiled with herbs, M meals/dishes, S soup, SP spore, P powder, T tablet

Chinese medicines have been utilized within several alternative medicinal practices to introduce positive changes in order to provide better contributions for health care while developing future medicinal products for diseases and for use in therapy (Lin et al. 2015; Han et al. 2017). However, there are challenges to the development of TCM

products, due in part to the nature of the therapeutic potential and the fact that the mechanisms of action are often unclear. Furthermore, the materials used often do not meet the requirements of quality control and standardisation (Wen et al. 2017), and hence they are difficult to register as prescription drugs. Many materials have not

<figure><img src="image_15.png" /></figure>
been tested thoroughly to the standards of western medi-
cine and can therefore only be sold as over-the-counter
drugs or even neutraceuticals. Many challenges still stand
in the way towards international collaborative exchange for
opportunities to develop TCM, thereby advancing it at the
global level (Chen et al. 2016). The use of mushrooms is
advantageous when compared with the plants used in
TCM, since mushrooms can be produced biotechnologi-
cally under controlled conditions and with standards,
making them easier to work with than species that have to
be grown and harvested in nature. In conclusion, to achieve
the global acceptance of TCM products, it will be neces-
sary to conduct clinical trials based on high quality stan-
dardised materials.

8. Cardiovascular disease control by fungi

Cardiovascular diseases include diseases of the heart,
vascular diseases of the brain, and diseases of blood vessels
(Mendis et al. 2011). Elevated levels of plasma cholesterol
are responsible for these diseases, as they play a major role
in atherosclerosis, the clogging or hardening of arteries
caused by accumulations of fatty deposits (usually
cholesterol) (Miller 2001). These diseases were responsible
for 17.3 million deaths per year in 2015, and are the leading
source of deaths worldwide (GBD 2015). Inhibition of de
novo synthesis of cholesterol was demonstrated to be an
effective method for reducing plasma cholesterol levels
(Miller 2001). The rate determining step is the reduction of
3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) to
mevalonate by HMG-CoA reductase; hence selective
inhibition of the latter enzyme, can reduce the synthesis of
cholesterol (Brown et al. 1976; Endo et al. 1976). The most
important class of HMG-CoA reductase inhibitors are the
statins (Fig. 8), which are derived from fungal natural
products and contain two types of structures moieties, a
hexahydro-naphthalene system and a β-hydroxylactone
system. The statins are also the best-selling class of phar-
maceutical drugs, with an annual turnover in the range of
US$50 billion. Therefore, the most important compounds
of this type are treated here.

<figure><img src="image_16.png" /><figcaption>**Fig. 8** Chemical structures of statins with cholesterol-lowering properties</figcaption></figure>

Brown et al. (1978) isolated compactin (22) (also known
as mevastatin and ML-236B) from Penicillium brevicom-
pactum as an antibiotic, but its hypocholesterolemic
activity was later revealed by Endo et al. (1976), who used
the name “ML-236B”. Later another HMG-CoA reductase
inhibitor named mevinolin (23) was isolated from Asper-
gillus terreus by Alberts et al. (1980). The first statin drug
approved by the United States Food and Drug Adminis-
tration was lovastatin (24) in 1987 (Jahromi et al. 2012).
Although lovastatin is produced by several species of
Penicillium (Endo et al. 1976), Monascus species (Miyake
et al. 2006; Sayyad et al. 2007), Doratomyces, Gymnoas-
cus, Hypomyces, Phoma and Trichoderma, the commer-
cialized product is derived from Aspergillus terreus
(Jahromi et al. 2012). Another statin containing a product
called Xuezhikang or “red yeast rice extract”, produced by
the fermentation of Monascus spp., has been widely used in
China for centuries for treating circulatory disorders. The
low density lipoprotein lowering ability of “red yeast rice
extract” is clinically proven and contains lovastatin (Lu
et al. 2008).

Solid state fermentation, or submerged cultures, can be
used for the production of lovastatin (Ruddiman and
Thomson 2001; Lai et al. 2003; Suryanarayan 2003; Wei
et al. 2007; Jaivel and Marimuthu 2010; Pansuriya and
Singhal 2010; Jahromi et al. 2012), but production is sig-
nificantly higher in the former (Jaivel and Marimuthu 2010;
Jahromi et al. 2012). Sorghum grain, wheat bran, rice and
corn are used as substrates for solid state fermentation (Wei
et al. 2007; Jaivel and Marimuthu 2010; Jahromi et al. 2012).

Basidiomycetes are a good source of nutrient supple-
ment for humans. Certain molecules in mushrooms can
modify cholesterol absorption, metabolism, and also
modulate the gene expression related to cholesterol
homeostasis (Gil-Ramírez et al. 2016). Grifola frondosa,
Hypsizigus marmoreus and Pleurotus ostreatus were able
to differentially modulate the gene expression patterns of
mice livers (Gil-Ramírez et al. 2016). Of the biologically
active compounds from fungi that can reduce the amounts
of cholesterol in the blood, the most studied are ergosterol
derivatives (Gil-Ramírez et al. 2016). The cholesterol-
lowering properties are mainly caused by the structural similarity with cholesterol (Gil-Ramírez et al. 2016). Further, the biological activity of β-glucans and chitin may be due to their binding abilities to cholesterol receptors (Gil-Ramírez et al. 2016).

Francia et al. (1999) recorded 16 species of edible mushrooms with biological activities against cardiovasculiar disease. Species of the genera Auricularia (Fan et al. 1989), Ganoderma (Kabir et al. 1988), Grifola (Kubo and Namba 1997), Pleurotus (Bobeck et al. 1991) and Tremella (Cheung 1996) have been reported to contain cholesterol-lowering compounds. Ophiocordyceps sinensis has also been shown to reduce total cholesterol levels, which has been attributed to the fact that it contains polysaccharide “CS-F30” composed of galactose, glucose and mannose (Kiho et al. 1996). For instance, low density lipoprotein cholesterol levels were reported to be reduced by Auricularia auricula-judae (Fan et al. 1989) and Tremella fuciformis (Cheung 1996), and triglyceride levels were reported to be reduced by Grifola frondosa (Kubo and Namba 1997), Lentinula edodes (Kabir and Kimura 1989) and Ophiocordyceps sinensis (Kiho et al. 1996).

## 9. Antiviral agents

Health and mortality-debilitating diseases caused by viruses continue to cause serious global epidemics, especially

in cases where vaccines and antiviral chemotherapies are insufficient or not available. The current state of virus-related pandemics is also significantly limiting drug efficacy by the emergence of drug-resistant strains. Hence, there is an urgent need to identify and develop natural product-inspired drug leads that could help control viral infections. A plethora of potentially active natural products have been isolated from fungi and screened for antiviral activity, even though none of them has reached the market yet. This entry focuses on natural products exhibiting potent activity on selected human pathogenic viruses, such as the human immunodeficiency virus (HIV), influenza virus, herpes simplex virus (HSV), hepatitis virus and other human pathogenic viruses such as enterovirus-71, and respiratory syncytial virus (RSV).

### Human Immunodeficiency Virus (HIV) inhibitory natural products from fungi

A comprehensive review of the literature identifies three main targets for anti-HIV drug discovery: virus entry, reverse transcription and integration.

The entry of HIV involves interactions with proteins and is a target for the discovery of new viral entry blockers. Examples of a few discoveries are provided. A bis-indolyl quinone, hinnuliquinone (25) (Fig. 9) from an unknown fungus isolated from Quercus coccifera, inhibited wild-type and clinically-resistant HIV-1 protease. HIV-1 protease is a key enzyme involved in the replication and

<figure><img src="image_17.png" /><figcaption>**Fig. 9** Chemical structures of fungal metabolites that were reported to possess antiviral activities</figcaption></figure>
maturation of the HIV-1 virus (Singh et al. 2004). Alter-
toxins I–III and V, oxidized perylenes from *Alternaria
tenuissima*, inhibited HIV-1 replication at micromolar
concentrations (Bashyal et al. 2014). The dimeric tetrahy-
droxanthone, penicillixanthone A from *Aspergillus fumi-
gatus* displayed strong anti-HIV activity by inhibiting
CCR5-tropic HIV-1 SF162 and CXCR4-tropic HIV NL4-3
(Tan et al. 2017). A marine-derived *A. niger* produced
malformin C, which exhibited a very strong anti-HIV-1
activity (Zhou et al. 2015a). An endophytic *Aspergillus* sp.
CPCC 400735 produced three phenalone and cytochalasin
derivatives also showing anti-HIV activity (Pang et al.
2017). Concentricolide from “*Daldinia concentrica*” (tax-
onomy doubtful since this species does not occur in China
according to the world monograph by Stadler et al. 2014)
inhibited HIV-1 by induction of cytopathic effects (Fang
and Liu 2009). Novel sesquiterpenoids from *Paraconio-
thyrium brasiliense* showed moderate anti-HIV-1 replica-
tion in C8166 cells (Liu et al. 2010b). The pupukeanane
sesquiterpenoid chloropupukeannolide A from *Pestalo-
tiopsis fici* showed significant anti-HIV-1 activity (Liu et al.
2010c). The cytochalasan perconiasin J and the meroter-
penoid periconone B from *Periconia* sp. displayed mod-
erate anti-HIV activity (Liu et al. 2016, 2017b). The
farnesylated isoindolinones stachybotrysams A–C and the
phenylspirodrimane derivatives stachbotrysin A and G
from *Stachybotrys chartatum* displayed moderate anti-HIV
activity (Zhao et al. 2017a, b).

The three consecutive functions controlled by HIV
reverse transcriptase are: RNA reverse transcription to
DNA, degradation of RNA template by RNase H, and
duplication of the remaining DNA strand. Inhibition of
these processes is important for the discovery of anti-HIV
drugs. Stachybosin D (26) (Fig. 9), a phenylspirodrimane
metabolite from a sponge-derived isolate of *Stachybotrys
chartarum*, showed inhibitory effects on HIV-1 replication
by targeting reverse transcriptase. It was able to inhibit
NNRTIs-resistant strains and wild-type HIV-1 (Ma et al.
2013).

Integrase is the only protein encoded by HIV-1, aside
from the enzymes protease and reverse transcriptase. Singh
et al. (1998, 2002a, b, 2003a, b, c) described several
compounds with inhibitory activity against integrase from
various fungal species. Accordingly, equisetin and pho-
masetin from *Fusarium heterosporum* and *Phoma* sp.,
respectively Singh et al. 1998); integracins (Integrastatin A
(27)) from *Cytonaema* sp. (Singh et al. 2002a); integras-
tatins (from an unidentified fungus; cf. Singh et al. 2002b);
epiphiobolins C and K from “Neosartorya”; i.e., *Asper-
gillus* sp.; 8-O-methylanthragallol from *Cylindrocarpon
ianthothele*; hispidin and caffeic acid from *Inonotus
tamaricis*; 3-hydroxyterphenyllin from *Aspergillus can-
didus* (Singh et al. 2003a); naphtho-γ-pyrones from

*Fusarium* sp. (Singh et al. 2003b); and xanthoviridicatins
from *Penicillium chrysogenum* (Singh et al. 2003c) all
showed low micromolar inhibition against the cleavage
reaction of HIV integrase. Funalenone from *Penicillium* sp.
FKI-1463 also had the same effect (Shiomi et al. 2005).

*Influenza virus inhibitory natural products from fungi*

The H1N1 and H3N2 viruses are among the targets of
natural products of fungal origin with anti-influenza
activity. The terpenoid stachyflin (28), isolated from a
marine-derived isolate of *Stachybotrys* showed modest
activity against the influenza A virus (H1N1) with an IC₅₀
of 3 × 10⁻³ µM (Minagawa et al. 2002). The γ-pyrone
isoasteltoxin from *Aspergillus ochraceopetaliformis*
showed low micromolar activity (IC₅₀ = 0.23 µM) against
both influenza viruses (Wang et al. 2016). Another
*Aspergillus* sp., strain produced the γ-pyrone derivative
asteltoxin E with IC₅₀ values of 6.2 and 3.5 µM against
H1N1 and H3N2, respectively (Tian et al. 2016). 3β-Hy-
droxysterol from *Pestalotiopsis* sp. (Sun et al. 2014) and
chermesinone from *Nigrospora* sp. (Zhang et al. 2016b)
also had moderate inhibitory effects. Aureonitol, a
metabolite of *Gliocladium* spp. inhibited influenza A and B
virus replication with an EC₅₀ of 100 nM against H3N2 via
suppression of influenza hemagglutination, while signifi-
cantly impairing viral adsorption (Sacramento et al. 2015).

*Herpes Simplex virus (HSV) inhibitory natural products from fungi*

The impotant human pathogenic viruses HSV-1 and HSV-2
were also subject of sceening programs of fungal
metabolite libraries, even though up to date no drug could
be discovered that would match the activity of the market
standard. Coccoquinone, an anthraquinone from *Aspergil-
lus versicolor*, demonstrated an IC₅₀ of 3 µM against HSV-
1 (Huang et al. 2017a, b). Five lipopeptides from the
marine-derived fungus *Scytalidium* sp. showed moderate
anti-HSV-1 and anti-HSV-2 activities in a dose- and time-
dependent pattern (Rowley et al. 2003). The diphenyl ether
glycoside cordyol C from *Cordyceps* sp. BCC 186 exhib-
ited significant anti-HSV-1 activity with an IC₅₀ value of
1.3 µg/ml (Bunyapaiboonsri et al. 2011).

*Hepatitis virus inhibitory natural products from fungi*

One novel tricyclic polyketide derived from a collection of
fungal-derived compounds, vanitaracin A (29), was
reported to inhibit viral entry process with an IC50 value of
0.6 µM and good selectivity. It was observed to directly
interact with the HBV entry receptor correlated to hepatitis
D virus and impaired viral bile acid transport pathway.
This compound also inhibited all HBV genotypes (A–D)
(Kaneko et al. 2015). The anthraquinone metabolite, 2'R-1-
hydroxyisorhodoptilonmetrin, showed better anti-hepatitis
B virus activity as compared to the positive drug control,
lamivudine (Jin et al. 2018). The epipolythiodioxopiperazine derivative, 11'-deoxyverticillin A, showed antiviral activity by decreasing HBV-X replication through inhibition of Akt activity or depletion of the autophagic genes, LC3 and p62 (Wu et al. 2015a). Sandargo et al. (2018) and Narmani et al. (2019) have recently discovered additional anti-HCV agents like 4-hydroxypleurogrisein (30) from the nematode trapping basidiomycete *Hohenbuehelia grisea* and cytosporaquinone B (31) from an Iranian phytopathogen belonging to the genus *Cytospora*. Recently Sandargo et al. (2019b) reported the meroterpenoid rhodatin (32) from cultures of the rare basidiomycete *Rhodotus palmatus* and also found significant anti-HCV activities for this compound, which features a new carbon skeleton.

In view of the fact that newly arising viral diseases are steadily being reported and they can spread more easily due to globalization effects, the search for novel antiviral agents is as of recently gaining importance. Some of the natural products pointed out in this entry may potentially find their way to antiviral drug development in the future. However, the discovery of new chemical derivatives with nanomolar activity and high selectivity indices is still warranted.

## 10. Immunosuppressive and immunomodulatory agents from fungi

Immunosuppression is a form of therapy that prevents the immune system of patients from acting against transplanted tissues and organs; without the availability of

immunosuppressive drugs, the progresses made in modern medicine, in particular regarding kidney, heart and liver transplants, would be unthinkable. Furthermore, immunosuppressants are used to control severe manifestations of allergic and autoimmune related diseases. Many of these drugs specifically address certain biochemical pathways that are crucial for the functioning of human defense against alien organisms, such as pathogens, by selectively inhibiting the immunocompetent lymphocytes or signal transduction cascades that regulate the transcription of cytokines. For this reason, patients treated with immunosuppressants often have to be hospitalized and treated in parallel with antibiotics to prevent infection.

Some of the most important immunosuppressive drugs are natural products that are being produced biotechnologically by fermentation of bacteria and fungi. While tacrolimus and sirolimus are derived from actinobacteria and will accordingly not be treated here, cyclosporine (35) and mycophenolate mofetil (33) are fungal metabolites, and the present entry is therefore dedicated to these important molecules (Fig. 10).

Mycophenolic acid (34) (Fig. 10) was the first antibiotic discovered and isolated in crystalline form from fungi, contrary to what is written elsewhere in the literature, where the penicillins are often regarded as the oldest natural antibiotics. Biogenetically, this compound is a meroterpenoid, produced by *Penicillium* species, including *P. brevicompactum* and *P. roquefortii*, and its biosynthesis has recently been elucidated in the latter species (Del-Cid et al. 2016). For various reasons, the compound never made

<figure><img src="image_18.png" /><figcaption>**Fig. 10** Chemical structures of immunosuppressive drugs derived from fungal fermentation</figcaption></figure>

<figure><img src="image_19.png" /></figure>
it into clinical development as an antibacterial or antifungal agent (Bentley 2000; Bills and Gloer 2016). Ultimately, its utility as an immunosuppressant became evident, and it is now the active principle of several marketed drugs, such as Myfortic® and CellCept®. The compound selectively inhibits inosine monophosphate dehydrogenase (IMPDH), an enzyme that is crucial for the biosynthesis of guanosine nucleotides in mammalian cells. As this enzyme is more essential in the T- and B-lymphocytes than in other cell types, and its isoform in the lymphocytes is more sensitive to mycophenolic acid, the drug has a more potent cytostatic effect on lymphocytes than on other cell types, and thereby suppresses the immune system (Allison and Eugui 2000). Another secondary beneficial effect of mycophenolic acid is the depletion of tetrahydrobiopterin, which is a co-factor for the inducible nitric oxide synthase (iNOS). Consequently, the administration of mycophenolic acid prevents damage of tissues mediated by peroxynitrite. The drug is mainly being used to prevent organ rejection following transplants, as well as in the therapy of psoriasis (Epinette et al. 1987) and other immunological disorders.

Cyclosporine A (35) (Fig. 10) was first discovered as a mildy active antifungal antibiotic by Dreyfuss et al. (1996), who also gave the first hints as to its immunomodulatory activity. The compound is a nonribosomally biosynthesized peptide derived from fermentation of the ascomycete *Tolypocladium inflatum*, and its biosynthetic gene cluster was recently elucidated by Bushley et al. (2013). After years of intensive research, it was found that this cyclopeptide has a highly specific biochemical mode of action as it selectively binds to cyclophilin A. This protein is an inhibitor of calcineurin, which is responsible for activation of transcription of the cytokine, interleukine 2. If interleukine 2 is depleted, the immune response of the human body will be suppressed and rejection of transplants can be prevented (Green et al. 1981; Wiesinger and Borel 1980). Therefore, cyclosporine A has become the active ingredient of blockbuster drugs, such as Sandimmune®, Neoral® and Restasis®). In addition to organ transplants, such immunosuppressants can be very useful in the therapy of other diseases, including allergies and neurodegeneration. An example for the latter indication is the synthetic drug, fingolimod (Gilenya®; 21 in Fig. 6), which is described further above in chapter 6.

In this context, it is worthwhile to note that many fungal metabolites are highly useful in therapy because they show the opposite activities in biological systems, i.e., they boost the immune system and therefore increase the resistance against pathogens or even help to prevent cancer. Striking examples for such molecules are the β-glucanes and protein–polysaccharide complexes that are being treated elsewhere herein.

## Strategies against plant disease

Fungi are important agents in combating various fungal pests and plant diseases found in greenhouses, the field, and even post-harvest. Fungi also have the potential to be used against certain animal parasites, such as nematodes. In this section, we discuss how fungi are being used to control plant disease, pests, nematodes, and herbicides, as well as their possible future applications.

## 11. Biocontrol of plant disease using endophytes

Fungal pathogens are the chief agent of plant disease, effecting severe agricultural losses worldwide (Hyde et al. 2014; Punja and Raj 2003; Strange and Scott 2005; Horbach et al. 2011). Agrochemicals play a significant role in plant disease management and ensure sustainable and productive agriculture systems. However, the intensive use of chemicals (determined by frequent and high dose of pesticides) has adverse effects on human health, ecosystem functioning, and agricultural sustainability (Anderson et al. 2004; Vinale et al. 2008; Suryanarayanan et al. 2016). Biocontrol is a strategy used to control plant pathogens, resulting in minimal impact to the environment (De Waard et al. 1993; Vinale et al. 2008).

Endophytes reside asymptomatically within a plant for at least part of their life cycle (Carrol 1998; Huang et al. 2009; Sun et al. 2011; Clay et al. 2016). Fungal endophytes can be broadly classified into two groups, the clavicipitaceous (C) and the non-clavicipitaceous (NC). These endophytes are classified based on evolutionary relatedness, taxonomy, host plant range and ecological function (Hyde and Soytong 2008; Rodriguez et al. 2009; O'Hanlon et al. 2012; Santangelo et al. 2015). Clavicipitaceous endophytes, including *Atkinsonella*, *Balansia*, *Balansiopsis*, *Echinodothis*, *Epichloë*, *Myriogenospora* and *Parapichloë* species are commonly associated with grasses in the family Poaceae and rely on their host throughout their life cycle as mutualist species (Rodriguez et al. 2009; Purahong and Hyde 2011; O'Hanlon et al. 2012; De Silva et al. 2016). Non-clavicipitaceous endophytes, such as *Fusarium* sp., *Colletotrichum* sp., *Phomopsis* sp. and *Xylaria* sp. are found in most terrestrial plants, and might not inhabit the host plants for their entire life cycle (Rodriguez et al. 2009; Delaye et al. 2013; De Silva et al. 2016; Jayawardena et al. 2016).

Endophytes are neutral or beneficial to their plant hosts (Backman and Sikora 2008). They boost host plant growth, fitness, stress tolerance, and alter interactions with pests and pathogens (Oono et al. 2015; Clay et al. 2016). Endophytes also provide protection against herbivory (O'Hanlon et al. 2012; Santangelo et al. 2015). More
importantly, endophytes have potential as an unexplored
source of candidate strains for potential biocontrol appli-
cations (Ek-Ramos et al. 2013; Oono et al. 2015). For
example, endophytic *Ampelomyces* species parasitize
powdery mildews (Busby et al. 2016). Since powdery
mildews are biotrophs, their antagonists act mainly through
antibiosis and mycoparasitism (Busby et al. 2016).

Biocontrol strategies utilize antagonistic mechanisms to disrupt the life cycle of pathogens (cf. Fig. 11), leading to the prevention of infection, reduction in colonization of host tissues, reductions in sporulation, and affecting the pathogen's ability to survive (Punja and Raj 2003; Busby et al. 2016). The hyperparasitic antagonism may be mediated by factors such as include the as production of lytic enzymes and/or antibiotics, while other biocontrol agents may induce the host plant's defense or just compete with the pathogen for nutrients and ecological niches (Yan et al. 2015; Busby et al. 2016; Lecomte et al. 2016; Schlegel et al. 2016).

Mejía et al. (2008) studied endophytes within the heal-
thy leaves of *Theobroma cacao*, as well as their antagonism
against the pathogenic Basidiomycota species *Monilioph*
*thora perniciosa* (witches broom), *Moniliophthora roreri*
(frosty pod rot) and the oomycete *Phytophthora palmivora*
(black pod rot). The results showed that two endophytes,
identified as *Colletotrichum gloeosporioides* and *Clonos-
tachys rosea*, respectively, decreased pod loss due to black
pod rot, and reduced sporulating lesions in cacao pods
caused by *Moniliophthora roreri*.

Endophytic fungi from various host plants have been
shown to be effective biocontrol agents, including *Al-
ternaria* sp. and *Cladosporium* sp., isolated from wheat
(Huang et al. 2016), and *Alternaria alternata*, isolated from
grapevine leaves (Zhang et al. 2017b). The use of antag-
onistic endophytes as biocontrol agents, such as *Tricho-
derma* and *Chaetomium*, present an attractive option for
management of certain plant diseases. It is important to
screen potential endophytes through in-vitro experiments
following field experiments under different environment
conditions. In future research using molecular technologies
(e.g., metagenomics), ecological dynamics are essential to
developing commercial biocontrol agents, as these con-
tribute to sustainable agriculture. It is critical to note that
the species isolated as endophytes from a certain host plant
may be pathogenic to other plants. Moreover, a careful risk
assessment that excludes the possibility for the overpro-
duction of mycotoxins is a mandatory prerequisite for
registrations of new biocontrol agents, regardless of whe-
ther the producer organisms are endophytes.

<figure><img src="image_20.png" /><figcaption>**Fig. 11** Methodology used to assess biocontrol activity of endophytes against pathogens in cocoa plants</figcaption></figure>

**12. Biocontrol of insects using fungi**

The loss in productivity due to crop damage from insects
represents a serious threat to the agricultural sector. The
global crop loss due to insects contributed to losses of
almost $470 billion each year (Culliney 2014), with the
global expenditures on pesticides being in the range of $56
billion in 2012. Strikingly, of the $56 billion, only $2–3
billion was spent on biopesticides (Marrone 2014). In the
USA, insecticides contributed to almost 14% of all pesti-
cide expenditures (Sabarwal et al. 2018).

The most common strategy for controlling insect inva-
sions is the use of synthetic chemical pesticides, such as
Chlorpyrifos, Acephate and Bifenthrin (Dai et al. 2019a).
However, insecticidal resistance has become an undeniable
phenomenon, and has led to the disastrous collapse of the
pest control in many countries (Naqqash et al. 2016). There
are also other concerns arising regarding the use of these
synthetic chemicals, namely with food safety, adverse
effects to non-target organisms—especially those
beneficial antagonists of insects—and the environmental impact associated with the use of harmful chemical compounds (Sandhu et al. 2017). The drawbacks to conventional insecticides spurred the search for potent and eco-friendly biocontrol agents.

Biological control agents offer more advantages than their chemical counterparts, since they are safe for other non-target organisms and infect only specific species, with long-term results on target pests (Sanda and Sunusi 2016). In particular, the entomopathogenic fungi, have the capacity to reduce or eradicate insect populations. Most fungi used for the control of insect pests are ascomycetes, which are usually found in the soil and can cause natural outbreaks on their own when environmental conditions are favorable. Some fungal strains have been developed into commercial products because of their ability to be mass produced (e.g. *Beauveria bassiana*, *Lecanicillium muscarium*, *Metarhizium anisopliae*). These can infect a wide range of insect hosts. (cf. Fig. 12). Specific fungal strains in commercial products target insect groups such as Coleoptera, Diptera, Hemiptera, Hymenoptera, Lepidoptera and Orthoptera (Dauda et al. 2018).

Some entomopathogenic fungi can also exist as plant endophytes in a variety of hosts. They can exhibit dual functions, acting against insects and plant pathogens, thus giving protection to plant hosts. Moreover, they can have additional roles in endophytism, plant disease antagonism,

growth promotion and rhizosphere colonization (Yun et al. 2017; Jaber and Ownley 2018).

Generally, the first mode of action of entomopathogenic fungi is to produce sticky spores to insure adhesion to the body of the host. The non-specific adhesion mechanism of the conidia is due to their hydrophobic properties, which has protein interactions with the hydrophobic exoskeleton of the susceptible host. The spores germinate quickly and initiate penetration of the insect exoskeleton. The fungal cells multiply in the hemocoel of the host's body, increasing the turgor pressure and eventually killing the insect. The entomopathogen grows in the host's cadaver to optimize spore production and dispersal under favorable environmental conditions (Roy et al. 2006). High numbers of spores are required to insure infection, with a minimum of $1 \times 10^8$ to $1 \times 10^9$ conidia/ml (Inglis et al. 2012).

Entomopathogenic fungi also produce secondary metabolites that can act as toxins with insecticidal effects. Proliferating protoplasts produce these compounds to weaken the host's defense mechanisms, causing rapid death (Hussain et al. 2014). The entomopathogens that produce toxins are more effective at killing the insect hosts as compared with those strains that do not produce such metabolites (Kershaw et al. 1999). There are a variety of active compounds produced by entomopathogenic fungi that exhibit insecticidal properties, as listed in Table 4. Beauvericin (36 in Fig. 13) is a well-known active compound produced by entomopathogenic fungi. It plays a key

<figure><img src="image_21.png" /><figcaption>**Fig. 12** Entomopathogens infecting insect hosts.</figcaption></figure>

a Acrodontium crateriforme,

b Cordyceps militaris,

c Ophiocordyceps nutans
role in the virulence of fungi that infect arthropods (Rohlf and Churchill 2011). It is a cyclic hexadepsipeptide, containing three D-hydroxyisovaleryl and three N-methylphenylalanyl residues in alternating sequence, and belongs to the enniatin antibiotic family. It is structurally similar to enniatins; however, it differs in the nature of its N-methylamino acid (Wang and Xu 2012). It was first isolated from *Beauveria bassiana* (Hamill et al. 1969) and later from *Fusarium* species (Liuzzi et al. 2017). Beauvericin has a strong insecticidal function against a broad spectrum of insects. Since the target insects are moving organisms, the entomopathogenic fungi producing beauvericin are more effective insecticidal agents than the direct use of the compound. Beauvericin was first discovered to have insecticidal activity by Hamill et al. (1969). Other studies proved the efficacy of beauvericin in killing other insects, such as *Calliphora erythrocephala*, *Lygus* spp. (Leland et al. 2005), *Aedes aegyptii* (Grove and Pople 1980), *Spodoptera frugiperda* and *Schizophagus graminum* (Ganassi et al. 2002); however, it can also be toxic to bees, thus posing a threat to other beneficial insects when applied in the field.

**Bassianolide (37)** a cyclic depsipeptide from *Lecanicillium lecanii*, exhibits moderate cytotoxicity and an immunosuppressive effect to insect hosts. It can cause significant maximum mortality to *Plutella xylostella* at 0.5 mg/ml concentrations (Keppanan et al. 2018). As in

other groups of fungal cyclopeptides, several variants if bassianolies are known (Matsuda et al. 2004).

**Destroxins (38–40)**, the cyclic hexadepsipeptide mycotoxins are produced by *Metarhizium anisopliae*. They can kill a variety of insect pests. The purified destruxins can cause toxic effects on the larval developmental stage of mosquitoes (*Aedes aegyptii*) with high mortality rates (Ravindran et al. 2016). The study of Dong et al. (2016) also revealed positive correlations between destruxin production and blastospore formation, and the producer strain has the potential to be developed into a mycoinsecticide.

**Enniatins (41–44)** are only produced by *Fusarium* species. They act as ionophores that bind with ammonium in the transport of ions in the lipid bilayer membrane of the cell. This ionophoric property of enniatins leads to the toxic action in the cell through the disturbance of the normal physiological concentration. Their best studied derivative, Enniatin B was previously shown to exhibit insecticidal activity against blowfly (*Calliphora erythrocephala*) and mosquito larvae (*Aedes aegypti*) (Grove and Pople 1980).

The successful application of entomopathogenic fungal strains relies on several factors, such as level of virulence, production efficiency and level of safety to humans and other non-target species. Virulence depends on a complex of factors, such as spore hydrophobicity, which is involved in the conidial adhesion, germination polarity of the spores wherein unidirectional spores are more virulent than multi-

<table><caption>**Table 4** Some insecticidal compounds produced by entomopathogenic fungi</caption><thead><tr><th>Entomopathogen</th><th>Insecticidal compounds</th><th>References</th></tr></thead><tbody><tr><td rowspan="5">Beauveria bassiana</td><td>Bassiacridin</td><td>Matsuda et al. (2004), Quesada-Moraga et al. (2004), Ohshiro et al. (2006), Heneghan et al. (2011), Fisch et al. (2011), Süssmuth et al. (2011)</td></tr><tr><td>Bassianin</td><td></td></tr><tr><td>Bassianolide</td><td></td></tr><tr><td>Beauvericins (36)</td><td></td></tr><tr><td>Beauverolides</td><td></td></tr><tr><td rowspan="3">Beauveria tenella</td><td>Beauverolides</td><td>Ohshiro et al. (2006), Namatame et al. (1999), Süssmuth et al. (2011)</td></tr><tr><td>Beauvericins (36)</td><td></td></tr><tr><td>Bassianolide (37)</td><td></td></tr><tr><td>Lecanicillium lecanii</td><td>Helvolic acid</td><td>Kildgaard et al. (2014), Liang et al. (2016)</td></tr><tr><td>Fusarium spp.; “Paecilomyces” (Isaria) fumosoroseus, Paecilomyces (Isaria) tenuipes</td><td>Beauvericins (36)</td><td></td></tr><tr><td>Isaria sp.</td><td>Beauverolides</td><td>Briggs and Atkins (1966), Isogai et al. (1978), Elsworth and Grove (1980), Grove (1980)</td></tr><tr><td>Metarhizium anisopliae</td><td>Destroxins (38–40)</td><td>Wang et al. (2012), Yoshida et al. (2014)</td></tr></tbody></table>

<figure><img src="image_22.png" /></figure>
<figure><img src="image_23.png" /><figcaption>**Fig. 13** Chemical structures of insecticidal compounds from fungi</figcaption></figure>

directional ones, presence of hydrolytic enzymes to breach the host's defense wall, and sensitivity to abiotic factors like temperature and humidity. Moreover, entomopathogenic fungi should only be considered for commercialization if they demonstrate high production efficiency, wherein the strains have minimum requirements for growth and can be grown and mass produced in solid substrate (Hussain et al. 2014), thus reducing production costs.

Certain techniques should be considered in order to increase the effectiveness of mycoinsecticides. Since insect larvae usually embed themselves into plant tissues or soil and do not feed on crops (Hall 1998), they do not typically stay in a specific location and therefore are very difficult to target. Spray application can be inefficient at this stage and depositing mycoinsectides over a field or crop may not be effective. Therefore, granular formulations which coat dry spores onto bran or grains, or the drying and fragmentation

of mycelium to hold spores in starch, can both be effective methods to treat insects in the field (Hall 1998). Adding the granular substances to mycoinsecticides increases the accuracy and efficiency of the spray.

Several tactics have been identified by Lacey and Kaya (2007) to increase the effectiveness of mycoinsecticides. Preventive applications could be inefficient because the residues are not long-lasting, and therefore they should be applied only when the target pest is seen. Mycoinsecticides should be applied before the pest population reaches peak numbers, and therefore early application is essential. Timing is also important: identifying the life cycle of the host, which has a higher probability of being in contact with the spores, could also increase the effectiveness of the mycoinsecticide. For example, *Beauveria bassiana* is more efficient in infecting active nymphs than winged adults. Moreover, mycoinsecticides should not be applied during
droughts because the environmental conditions are not favorable for the germination of the spores.

Approximately 750 fungal species have been identified as insect pathogens; however, in 1998 only 12 species were utilized as mycoinsecticides in insect sprays (Wraight and Carruthers 1998). By 2007 the number of mycoinsecticides had increased to about 110 commercially available products in the market (de Faria and Wraight 2007). The number has continued to increase, with almost 2700 arthropod pesticides introduced worldwide (Cock et al. 2010), in which about 230 species of biological control agents have been marketed and available for commercial use (van Lenteren 2012). The demand and commercial application of microbial pesticides have increased tremendously. Of 82 microbial biopesticides registered in Brazil, nearly 46% are mycoinsecticides (Mascarin et al. 2018). Fungal biopesticides contributed to a large portion of this increased demand and popularity in pest management in the past two decades (Jaronski and Mascarin 2017). Several products have already disappeared from the international marketplace because they were not commercially successful, but a substantial number of fungal species have been exploited (Kabaluk et al. 2010; Lacey et al. 2015) (Fig. 12).

De Faria and Wraight (2007) and Mascarin et al. (2018) listed representatives of mycoinsecticides derived from *Beauveria*, *Isaria*, *Lecanicillium*, and *Metarhizium* species found in the market. Most developed mycoinsecticides were made for European and American markets, whereas only a few were aimed at the African and Asian markets (Table 5).

There have been continual obstacles and challenges to the development and commercialization of fungal biological control agents for use in controlling insect populations, ranging from gaps in understanding the basic biological knowledge to their potential socio-economic impact. Problems regarding the effectiveness of insecticides are linked to economic production. The increase in pest-resistance to chemical formulas has led to drastic environmental problems. New chemical insecticides are not being developed quickly enough, and therefore there is room for microbial insecticides, which are growing at the rapid pace of 10–25% per year (Starnes et al. 1993), within the market. Enhancing the virulence of potential fungal species is essential for mycoinsecticides to develop and reach their market potential.

The future of mycoinsecticides is promising, with continued research to increase pathogenic virulence of entomopathogenic fungi in order to achieve commercial success with genetic and physiological engineering. Further studies should be conducted to determine the fungal traits responsible for the effectiveness of mycoinsecticides,

enhancement of their virulence, and development of eco-friendly and effective pest management strategies.

## 13. Biocontrol of nematodes and fungal nematicides

Plant-parasitic nematodes are parasites which cause severe damage to many economically important crops such as tomatoes, potatoes and wheat. For example, around US $80 billion of yield losses are caused annually by damage from plant root-knot nematodes, such as *M. javanica* and *M. incognita* (Li et al. 2007). Nematicidal chemicals that were once rather effective, such as methyl bromide, have ultimately been banned because they are broad-spectrum biocides that kill all life in the soil and contribute to the depletion of the ozone layer, thereby causing grave problems to the environment. Therefore, in recent years there have been great efforts in both academia and industry to find ecologically viable alternatives.

Nematophagous fungi are capable of controlling plant parasitic nematodes through antagonistic behaviour (Zaki and Siddiqui 1996). There are more than 700 species of nematophagous fungi, which are found in fungal taxa including Mucoromycota, Basidiomycota, Ascomycota and Chytridiomycota (Li et al. 2005; Degenkolb and Vilcinskas 2016). These nematophagous fungi are categorized into four groups according to their mode of parasitism: nematode-trapping (otherwise known as predacious); nematode egg and female parasites; endoparasitic; and toxin-producing fungi (Li et al. 2005; Degenkolb and Vilcinskas 2016). Nematode-trapping fungi form specific trapping structures on hypha, such as adhesive knobs, constricting rings and adhesive networks; these three trapping devices can be categorized further into seven types: simple adhesive branches, unstalked adhesive knobs, stalked adhesive knobs, non-constricting rings, constricting rings, two dimensional networks and three-dimensional networks (Rubner 1996).

The constricting ring trap is the most sophisticated morphological adaptation, which can be found in fungi that are no accommodated in the genus *Drechslerella* (Baral et al. 2018). When a nematode enters the ring, the three cells swell, forming three sphaerical structures that trap and immobilize the nematode (Jansson and Lopez-Llorca 2004). Endoparasitic fungi attack nematodes orally or by penetration of spores or zoospores through the cuticles of the nematode host (Moosavi et al. 2011). After infection, the hyphae develop inside the nematode and digests its content. For example, the spores of *Catenaria anguillulae* are ingested by sedentary nematodes such as *Heterodera spp.* The spores germinate in the esophagus and the mycelia then digest the nematodes; 10–12 h after the infection, zoosporangia develop to release new zoospores
<table><caption>**Table 5** Representative mycoinsecticides in the market (de Faria and Wraft 2007; Mascarin et al. 2018)</caption><thead><tr><th>Fungal species</th><th>Marketed trade name</th><th>Country registered</th><th>Pests targeted</th><th>Manufacturer</th></tr></thead><tbody><tr><td rowspan="14">Metarhizium anisopliae</td><td>Arizium</td><td>Brazil</td><td>Mahanarva fimbriolata, Notozulia (Zulia) entreriana</td><td>TecniControl</td></tr><tr><td>BioMetha GR Plus</td><td>Brazil</td><td>M. fimbriolata, Notozulia sp., Deois sp.</td><td>Biotech</td></tr><tr><td>Bioninsect</td><td>Brazil</td><td>M. fimbriolata, N. entreriana, D. flavopicta</td><td>Koppert do Brasil</td></tr><tr><td>GR-INN</td><td>Brazil</td><td>M. fimbriolata, N. entreriana, D. flavopicta</td><td>Agrivalle</td></tr><tr><td>Metamax Líquido</td><td>Brazil</td><td>M. fimbriolata</td><td>Bio Soja</td></tr><tr><td>Metapremium</td><td>Brazil</td><td>M. fimbriolata, N. entreriana, D. flavopicta</td><td>Biopremium</td></tr><tr><td>Metarfito</td><td>Brazil</td><td>M. fimbriolata, N. entreriana</td><td>Fitoagro Controle Biológico</td></tr><tr><td>Metarhizium JCO WP</td><td>Brazil</td><td>M. fimbriolata, N. entreriana, D. flavopicta</td><td>JCO</td></tr><tr><td>Metarhizium Oligos</td><td>Brazil</td><td>M. fimbriolata, N. entreriana, D. flavopicta</td><td>Oligos Biotecnologia</td></tr><tr><td>Metarhizium Probio</td><td>Brazil</td><td>M. fimbriolata, N. entreriana, D. flavopicta</td><td>Probio</td></tr><tr><td>MethaControl</td><td>Brazil</td><td>M. fimbriolata, N. entreriana, D. flavopicta</td><td>Simbiose</td></tr><tr><td>Methamax EC</td><td>Brazil</td><td>M. fimbriolata</td><td>Novozymes</td></tr><tr><td>Metie</td><td>Brazil</td><td>N. entreriana</td><td>Ballagro</td></tr><tr><td>Opala</td><td>Brazil</td><td>M. fimbriolata, N. entreriana</td><td>Lab. Biocontrole Farroupilha</td></tr><tr><td>Real Metarhizium</td><td>Kenya, South Africa, Ethiopia, Ghana</td><td>Mealybugs, thrips, whiteflies</td><td>Real IPM (Kenya)</td></tr><tr><td rowspan="3">Metarhizium brunneum</td><td>Met52 EC</td><td>USA</td><td>Whiteflies, thrips, weevils</td><td>Novozymes Biologicals (USA)</td></tr><tr><td>Met52 Granular</td><td>USA, Europe</td><td>Weevils, thrips</td><td>Novozymes Biologicals (USA)</td></tr><tr><td>Bio1020</td><td>Netherlands</td><td>Weevils</td><td>Novozymes Biologicals France</td></tr><tr><td rowspan="14">Beauveria bassiana</td><td>Adral</td><td>Colombia</td><td>Aphids</td><td>Bio-Crop (Colombia)</td></tr><tr><td>Agrivalle AUIN</td><td>Brazil</td><td>Bemisia tabaci, Cosmopolites sordidus, Dalbulus maidis, Tetranychus urticae</td><td>Agrivalle</td></tr><tr><td>Ballvéria</td><td>Brazil</td><td>B. tabaci</td><td>Ballagro</td></tr><tr><td>Bassicore SC</td><td>Colombia</td><td>Whiteflies</td><td>Core Biotechnology (Colombia)</td></tr><tr><td>BeauveControl</td><td>Brazil</td><td>B. tabaci, D. maidis, T. urticae</td><td>Simbiose</td></tr><tr><td>Beauveria JCO</td><td>Brazil</td><td>B. tabaci, C. sordidus, D. maidis, T. urticae</td><td>JCO</td></tr><tr><td>BioExpert</td><td>Colombia</td><td>Whiteflies, thrips</td><td>Live Systems Technology (Colombia)</td></tr><tr><td>Bioveria WP</td><td>Brazil</td><td>B. tabaci, C. sordidus, D. maidis, T. urticae</td><td>Bioenergia do Brasil SA</td></tr><tr><td>Bovemax EC</td><td>Brazil</td><td>H. hampei, Diaphorina citri, Hedypathes betulinus</td><td>Novozymes</td></tr><tr><td>Boveril WP</td><td>Brazil</td><td>B. tabaci, Hypothenemus hampei, Gonipterus scutellatus, T. urticae</td><td>Koppert do Brasil</td></tr><tr><td>Broadband</td><td>South Africa</td><td>Whiteflies, thrips</td><td>BASF South Africa</td></tr><tr><td>Cadete SC</td><td>Colombia</td><td>Whiteflies, thrips</td><td>Mycros Internacional (Colombia)</td></tr><tr><td>Ecobass</td><td>Brazil</td><td>B. tabaci, C. sordidus, D. maidis, T. urticae</td><td>Toyobo do Brasil</td></tr></tbody></table>
<table><caption>**Table 5** continued</caption><thead><tr><th>Fungal species</th><th>Marketed trade name</th><th>Country registered</th><th>Pests targeted</th><th>Manufacturer</th></tr></thead><tbody><tr><td>Isaria fumosorosea</td><td>Granada</td><td>Brazil</td><td>B. tabaci, C. sordidus, D. maidis, T. urticae</td><td>Lab. Biocontrole Farroupilha</td></tr><tr><td>Isaria javanica</td><td>Challenger</td><td>Brazil</td><td>Diaphorina citri</td><td>Koppert do Brasil</td></tr><tr><td rowspan="2">Isaria spp.</td><td>PFR-97 20% WDG</td><td>USA, Mexico</td><td>Whiteflies, aphids, thrips, mealybugs,</td><td>Certis, Inc. (USA)</td></tr><tr><td>PreFeRal WG</td><td>Europe</td><td>Whiteflies</td><td>Biobest (Belgium)</td></tr><tr><td rowspan="3">Lecanicillium muscarium</td><td>Nofly</td><td>USA</td><td>Whiteflies, aphids, thrips, psyllids,</td><td>Novozymes Biologicals (USA)</td></tr><tr><td>Pae-Sin</td><td>Mexico</td><td>Whiteflies</td><td>Agrobiológicos del Noroeste –</td></tr><tr><td>Successor SC</td><td>Colombia</td><td>Whiteflies, aphids, thrips</td><td>Live Systems Technology (Colombia)</td></tr><tr><td>Lecanicillium spp.</td><td>Mycotal</td><td>Europe, Japan</td><td>Whiteflies, thrips</td><td>Koppert Biological Systems</td></tr><tr><td rowspan="3">Lecanicillium spp.</td><td>Verzam</td><td>Honduras, El Salvador, Nicaragua, Jamaica</td><td>Whiteflies, aphids, thrips</td><td>Escuela Agrícola Panamericana</td></tr><tr><td>Vercani WP</td><td>Colombia</td><td>Whiteflies</td><td>Natural Control (Colombia)</td></tr><tr><td>Lecafol</td><td>Uruguay</td><td>Whiteflies</td><td>Lage y Cía. S.A., Uruguay</td></tr></tbody></table>

(Mankau 1980). The motile zoospores are adapted by having positive tropisms toward nematodes (Kumar et al. 2017). Nematode egg parasitic fungi play a key role in infecting the eggs of nematodes on two levels. Some fungi directly infect the nematode eggs by penetrating the egg-shell, while others indirectly affect the content of eggs, such as larvae or embryos (Jansson and Lopez-Llorca 2004). Recent studies found that *Ijuhya vitellina* forms hyphae from the infected egg shells of the cereal cyst nematode *Heterodera filipjevi* to develop into microscle-rotia (Ashrafi et al. 2017a). *Monocillium gamsii* and *M. bulbillosum* are two nematode-associated fungi parasitic to eggs of *H. filipjevi* (Ashrafi et al. 2017b). The first record of dark septate endophytes with nematicidal effects was reported from the new genus *Polyphilus*, represented by two new species (Ashrafi et al. 2018). Actually *Polyphilus* spp. have been isolated from nematode eggs as well as from healthy plant material.

Several studies related to nematode parasitic fungi that are specific to economically important crops have been conducted (Table 7). *Cochliobolus sativus* (on wheat and barley), *Dendriphiopsis* spp. (on tomato plants) and *Drechmeria coniospora* are some controlling agents for root knot disease caused by nematodes on wide variety of crops, especially tomatoes (Jansson et al. 1985). A combination of *Trichoderma* species and nematode-trapping

fungi was most effective in controlling plant-parasitic nematodes through egg parasitism (Szabo 2014). Moreover, there are some fungi which are capable of controlling nematode diseases in animals (Zhang et al. 2007). For example, animals infected by plant-parasitic nematodes are fed with fungal mycelium containing chlamydospores of nematode trapping fungi, e.g. *Duddingtonia flagrans* (Zhang et al. 2007). When fed to the animals, these spores produce traps in the faeces and surrounding grasses to capture newly hatched nematode juveniles (Zhang et al. 2007). *Purpureocillium lilacinum* is capable of controlling root-knot nematodes such as *M. javanica* and *M. incognita* on tomato, eggplant and other vegetable crops (Moosavi 2014). Some studies have reported the antagonistic behaviour of *Arthrobotrys dactyloides* against root knot nematodes on tomato plants, but other experiments with *Metacordyceps chlamydosporia* did not significantly reduce nematode population (Nordbring-Hertz et al. 2011). *Cochliobolus sativus* was also reported to be an effective biological control agent in controlling nematodes in Botswana (Mubyana-John and Wright 2011). Table 6 is a synopsis of nematode parasitic fungi, and the unique trapping mechanisms of nematode-trapping fungi. The combination of *Purpureocillium lilacinum* (previously referred to as “Paecilomyces lilacinus”) and *Dactylella lysipaga* was experimentally proved to be antagonistic to
<table><caption>**Table 6** Taxonomy of nematode parasitic fungi, their trapping mechanisms and parasitic nematodes.. Source Zhang et al. (2014a, b)</caption><thead><tr><th>Nematophagous fungal group</th><th>Phyla</th><th>Fungal name</th><th>Trapping structures</th></tr></thead><tbody><tr><td rowspan="6">Nematode-trapping</td><td rowspan="2">Zoophagomycota</td><td>Stylopage</td><td>Adhesive hyphae</td></tr><tr><td>Cystopage</td><td>Adhesive hyphae</td></tr><tr><td rowspan="4">Ascomycota</td><td>Arthrobotrys/Orbilia</td><td>Adhesive networks</td></tr><tr><td>Dactylellina/Orbilia</td><td></td></tr><tr><td>Drechslerella/Orbilia</td><td>Adhesive knobs and/or non constricting rings</td></tr><tr><td>Gamsylella/Orbilia</td><td>Constricting rings</td></tr><tr><td rowspan="2">Basidiomycota</td><td>Hohenbuehelia</td><td>Adhesive branches or unstalked knobs</td></tr><tr><td></td><td>Adhesive “hour-glass” knobs</td></tr><tr><td rowspan="6">Endoparasitic</td><td>Chytridiomycota</td><td>Myzocytiopsis</td><td>Zoospores</td></tr><tr><td rowspan="2">Blastocladiomycota</td><td>Haptoglossa</td><td>“Gun cells”, injection</td></tr><tr><td>Catenaria</td><td></td></tr><tr><td rowspan="4">Ascomycota</td><td>Harposporium/Podocrella</td><td>Zoospores</td></tr><tr><td>Meria</td><td>Ingested conidia</td></tr><tr><td>Haptocillium/Cordyceps</td><td>Adhesive conidia</td></tr><tr><td>Hirsutella</td><td>Adhesive conidia</td></tr><tr><td rowspan="4">Toxin-producing</td><td>Basidiomycota</td><td>Nematoctonus/Hohenbuehelia</td><td>Adhesive “hour-glass” knobs</td></tr><tr><td rowspan="3">Basidiomycota</td><td>Coprinus</td><td>Toxic droplets</td></tr><tr><td>Pleurotus</td><td>Toxin, spiny structures</td></tr><tr><td>(Omphalotus)</td><td></td></tr></tbody></table>

the root-knot nematode *Meloidogyne javanica* as well as the cereal cyst nematodes *Heterodera avenae* and *Radopholus similis* on tomato barley and banana plants (Zhang et al. 2014a, b). In another study, the combination of *Purpureocillium lilacinum* and *Monacrosporium lysipagum* was shown to be antagonistic to *Meloidogyne javanica*, the cereal cyst nematode (Zhang et al. 2014a, b).

Few studies conducted thus far have proven the efficiency of nematode parasitic fungi; however, individually these fungi do not possess all the desirable characters required to serve as high potential nematode control agents. Thus combinations of nematophagous fungi can be more effective, as these combinations are capable of being parasitic in all stages of the nematode life cycle. Therefore, it is necessary to develop new techniques to facilitate research on nematode parasitic fungi, especially for tracing high potential strains in the environment. More studies are needed to improve and introduce more efficient strategies to produce biological control agents without harming non-targeted nematode species.

Liquid fermentation and solid fermentation are two methods of mass producing nematophagous fungi. Solid culturing is preferable for fungi that cannot produce spores in liquids (Zhang et al. 2014a, b). However, liquid culturing is widely used for the mass production of the spores and mycelium of targeted fungi (Zhang et al. 2014a, b). In fact, the combination of these two methods enhances the effectiveness of the mass production of fungi, as the liquid method can be used for producing mycelia and the solid

method for producing conidia. The formulation of the fungal production is important in its commercialization as a bio control agent. Formulations are powders, wettable powders, emulsions, oil solutions, granular formulations, blending agents and microcapsules (Liu and Li 2004). Microencapsulation is a new method of commercializing bio control fungi (Patel et al. 2011). The latter researchers developed a novel capsule system using *Hirsutella rhossiliensis*. Jin and Custis (2011) also introduced a modified method for microencapsulation of *Trichoderma* conidia using sugar, which was then developed as a sprayable formulation.

Toxin-producing fungi secrete nematicidal metabolites to attack and immobilise nematodes before the development of hyphae inside the nematode (Degenkolb and Vilkinskas 2016). For example, *Purpureocillium lilacinum* produces acetic acid to immobilize juvenile parasitic nematodes (Djian et al. 1991). According to previous studies, more than 270 toxin-producing fungi have been recorded, including 230 nematicidal toxic compounds (Zhang et al. 2011a; Li et al. 2007). Recent examples include the report on the new genus and species *Pseudobambusicola thailandica*, which can control nematodes using its secondary metabolites, such as monocerin and deoxyphomalone (Rupcic et al. 2018). Linoleic acid is one of the nematicidal compounds that can be isolated from many fungi, including *Arthrobotrys* species, in which the production of this compound increases with the number of traps (Anke et al. 1995). *Pleurotus pulmonarius* and
*Hericium coralloides* are two basidiomycetes that exhibit strong nematicidal effect against *Caenorhabditis elegans*. p-Anisaldehyde (46) and other aromatic metabolites, as well as fatty acids, were identified as active principles (Stadler et al. 1994) (Fig. 14).

Metabolites with moderate nematicidal activity have been recently reported from a *Sanghuangporus* sp. collected in Kenya. In addition to 3, 14'-bihispidinyl and hispidin and the new dcerivative phelligridin L (49), with moderate effects on *Caenorhabditis elegans* (Chepkirui et al. 2018). Ophiotine (45), a depsipeptide from a species of the Phaeosphariaceae isolated from nemtatode eggs, was reported to have a moderate nematicidal effect on *Heterodera filipjevi* (Helaly et al. 2018a). *Chaetomium globosum* produces flavipin, which inhibits egg hatching and juvenile mobility of the root-knot nematode (*Meloidogyne incognita*) and the hatching of soybean cysts (*Heterodera glycines*) (Nitao et al. 2002). Chaetoglobosin A and its derivate 19-O-acetylchaetoglobosin A are two other recently demonstrated chemical compounds isolated from *Ijuhya vitellina*, a parasitite of the eggs of *Heterodera filipjevi* (Ashrafi et al. 2017a, b). However, the most important fungal nematicides found to date are (a) PF-1022A, a precursor of the semisynthetic drug emodepside (48), which is used in veterinary medicine (Jeschke et al. 2005); and (b) the cyclopeptide omphalotin A (47) from cultures of the basidiomycete genus *Omphalotus*, which was also developed as a nematicide against *Meloidogyne*, but did not yet make it to the market because of unfavourable costs of goods (Sandargo et al. 2019a).

Many studies have shown the parasitic efficiency of nematode parasitic fungi, but it is necessary to develop IPM strategies to optimize the ability nematophagous fungi to colonize plant roots. High virulence strains and

<figure><img src="image_24.png" /><figcaption>**Fig. 14** Chemical structures of fungal metabolites with nematicidal activity</figcaption></figure>
<figure><img src="image_25.png" /><figcaption>**Table 7** Commercial biological nematicides based on nematophagous fungi.. *Source* Zhang et al. (2014a, b), updated by using the current taxonomy</figcaption></figure>

<table><thead><tr><td>Products</td><td>Fungi</td><td>Modes of action</td><td>Nematode targets</td><td>References</td></tr></thead><tbody><tr><td>Bicon</td><td>Purpureocillium lilacinum</td><td>Egg and female parasitism</td><td>Unspecified</td><td>Poinar and Georgis (1994)</td></tr><tr><td>Miexianning</td><td>Purpureocillium lilacinum</td><td>Egg and female parasitism</td><td>Root-knot nematodes parasitizing tobacco</td><td>Shuhua et al. (2002), Zhou and Mo (2002)</td></tr><tr><td>Nemout</td><td>Unspecified NTF<sup>a</sup></td><td>Trapping</td><td>Meloidogyne javanica</td><td>Al-Hazmi et al. (1982), Ibrahim (1994), Williams et al. (1999)</td></tr><tr><td>Paecil/Bioact</td><td>Purpureocillium lilacinum</td><td>Egg and female parasitism</td><td>Unspecified</td><td>Holland et al. (1999)</td></tr><tr><td>Royal 350</td><td>Arthrobotrys irregularis</td><td>Trapping</td><td>Meloidogyne spp.</td><td>Cayrol et al. (1978)</td></tr><tr><td>Xianchongbike</td><td>Metacordyceps chlamydosporia</td><td>Egg and female parasitism</td><td>Root-knot nematodes<br>Parasitizing tobacco, peanut, soybean and watermelon</td><td>Zhou and Mo (2002)</td></tr></tbody></table>

formulations are important for the development of more efficient commercial products. The utilization of the secondary metabolites of fungi as nematicides is not satisfactory at the commercial level as there are few bionematicides available in the market. On the other hand, integrated nematode management strategies can be implemented to improve the effectiveness of nematicidal action and minimize the use of chemical nematicides to the soil. Future bio products should be targeted not only for one

species of nematode, but to disease complexes in concert with other pathogens like fungi, bacteria and viruses.

## 14. Biocontrol of weeds and herbicides from fungi

Weeds are plants that grow in a location where their presence has become undesirable due to their adverse effects on the ecosystem or human activities (Norris 1992; Kent 1994; Gadermaier et al. 2014). This is mainly due to
the fact that when a plant is introduced to a new environ-
ment without the influence of their natural enemies they
can grow without any obstruction and harm the native
population by taking the form of a weed (Kendrick 1992).
Thus, weeds can cause problems not only to the crop yield
and quality, but also to the nature by invading native
environment and its species composition. In order to ensure
the balance of ecosystems, global crop production and food
security; weed management is an important factor to be
considered (Bjawa 2014).

During the early 1990s weed management was domi-
nated by mechanical methods (Sommer 1996). The
importance of mechanical weed control has become limited
as it causes soil erosion and nutrient losses (Zimdahl 1993).
Thus, chemical herbicides became the viable alternative.
However, due to continuous use of the same herbicide with
the same mode of action, herbicide resistance increased
and for the last 20 years no chemical has been manufac-
tured with a different mode of action than the previous
products (Duke 2012). Researchers also identified the
herbicidal toxic impact on non-target soil organisms which
play an important role in degrading and decomposing
organic matter (Subhani et al. 2015; Zain et al. 2013).
Therefore, the need for biological weed control as well as
development of bio-herbicides has become important.

Biological control of weeds (see the diagram in Fig. 15
for an example) is an effective and efficient alternative
control method (Senthilkumar 2007). Biological weed
control is used against invading plant species that pose a
threat to endangered ecosystems and is designed to reduce
the competition for nutrients and space by weeds. The aim
of biological control of weeds is to decrease, suppress or
kill the weed population using host specific insect herbi-
vores or plant pathogens such as fungi, bacteria and viruses
(Bailey et al. 2010).

The initial concept for using fungal pathogens in weed
control was observed by a farmer in 1890 in the USA with
the thistle population controlled by a rust fungus (Wilson
1969). A similar situation was recorded by Morris (1991)
who observed that the Australian gall-forming rust fungus
*Uromycladium tepperianum* helps to reduce the infestation
of *Acacia saligna* at over 50 localities in South Africa.
Until 1970 biocontrol of weeds was not considered as a
solution or alternative to the massive use of chemical
herbicides (Pointing and Hyde 2001).

Biological control refers to the planned introduction of
an exotic bio control agent for permanent establishment for
long term control in an area where weeds are problematic
to the natural habitat (Evans 1998; Eilenberg et al. 2001).
Fair examples for using fungi in biocontrol of weed are the
successful use of the European rust fungus *Phragmidium
violaceum* to control European blackberry (*Rubus* sp.) in
Chile, or the use of *Puccinia chondrillina* to control

*Chondrilla juncea* (rush skeleton weed) in Australia, which
is considered as the most remarkable successes ever
achieved with biocontrol (Kendrick 1992). These rust fungi
are obligate biotrophs and cannot mass produce spores (or
even grow) in an artificial medium. Therefore, small
amounts of natural inoculum were introduced to the area
and the weed control was obtained by natural spore pro-
duction and dispersal (Cullen et al. 1973; Cullen and
Delfosse 1985; Kendrick 1992; Espiau et al. 1998).

Inocula of plant pathogens, applied to weeds in a similar
manner to synthetic herbicides are called bioherbicides.
When fungi are involved, they are referred to as mycoher-
bicides (Boyetchko et al. 1996). These pathogens usually
occur naturally on weeds in localities where they need to be
controlled, but not in sufficient amounts. Therefore, fungal
inocula are mass produced and sprayed on to the weeds.
These mycoherbicide products must undergo a government-
regulated registration process similar to chemical herbicides
before they are released for public use (Evans 1998).

Mycoherbicide studies began in the 1940s in several
countries with the aim to spread indigenous pathogenic
fungal species on target weeds as a control measure (Pointing
and Hyde 2001). In 1949, an attempt to control prickly pear
cactus using *Fusarium oxysporum* was unsuccessful (Julien
and Griffiths 1998). Kendrick (1992) mentioned another few
examples where mass produced fungal propagules are
applied as a mycoherbicides. A *Colletotrichum gloeospori-
oides* spore suspension was used to control northern joint
vetch (*Aeschynomene virginica*) in rice and soybean fields in
the USA. This was the first practical use of a fungus as a
mycoherbicide that was later commercialised as “Collego”.
Another example was water hyacinth, considered to be the
worst aquatic weed in the world, but could be controlled by
*Acremonium zonatum* and *Cercospora rodmanii*. However,
during the 1950s, spores of *Alternaria cuscutacidae* were
mass produced and applied to a dodder (*Cuscuta* sp.) (Wilson
1969, Julien and Griffiths. 1998). In 1963 China developed a
mycoherbicide for dodder, using *Colletotrichum gloeospo-
rioides* cf. *cuscutae* (*Colletotrichum cuscutae*) (Auld 1997;
Kendrick 1992).

In the 5th edition of “A world catalogue of agents and
their target weeds” by Winston et al. (2014), seven fungal
species are mentioned that were developed as mycoherbi-
cides and some of these are available as commercial
products. According to Walker and Connick (1983) and
Auld (1993) dew and temperature are the most important
factors for primary infection of pathogen in order to obtain
a successful disease development on the target weed. Some
researchers have insisted that it is important to consider the
time for secondary infection; which lead to a successive
distribution of disease in the field (Boyette et al. (1979).
According to Hasan and Ayres (1990) and El Morsy
(2004), *Stagnospora* species on *Calystegia sepium* required
only 3 weeks for control and *Alternaria alternata* on water
hyacinth took 2 months.

Finding a new active fungal isolate for mycoherbicide
production is not an easy task. There are a few important
factors that need to be considered before mass production
(O'Connell and Zoschke 1996). Mass availability of pro-
duct, scientific testing for laboratory and field conditions,
registration and commercializing procedures are necessary
(Evans 1998). Within the past three decades research for
improving this technology has increased. Even though the
target weeds and phyto-pathogenic species identifications
are still ongoing, interesting discoveries have surfaced
overtime. According to Gan et al. (2013), the number of
candidate genes found from the genomes of *Colletotrichum
gloeosporioides* and *Colletotrichum orbiculare*, which
were predicted to be involved in pathogenesis, showed the
potential to be exploited as mycoherbcides. The discovery
of genes in these two organisms related to the production of
Indole acetic acid (IAA), a component of some of the well
established herbicides, (see also chapter 21) showed that
these can be converted to mycoherbicides (Gan et al.
2013). Some *Phoma* species were also considered as a
successful candidate for the biocontrol of weeds. The
ability of *Phoma macrostoma* to inhibit the growth of dicot
plants was studied (Bailey et al. 2011, 2013; Smith et al.
2015). This fungus was used to control broadleaf weeds in
turf systems in Canada and the USA. A registered com-
mercial product of *Phoma macrostoma* is also available in
Canada and USA (Evans et al. 2013).

The secondary metabolites produced by some fungi
have been shown to have herbicidal activity. Castro de
Souza et al. (2016) found several *Diaporthe* spp. from the
Brazilian Pampa biome that have the ability to produce
secondary metabolites with herbicidal activity. The genus
*Diaporthe* is very rich in secondary metabolites, as recently
summarised by Chepkirui and Stadler (2017).

Biological control may take several years to take effect
and the effectiveness is influenced by a number of factors,
such as climatic conditions, geographical region and
management practices (Pointing and Hyde 2001). Among
these, high initial cost, limited number of natural enemies
and uncontrollable dissemination of biological control
agents after its release in nature are considered as disad-
vantages (El-Sayed 2005). Biological control is particularly
useful in areas where other conventional control methods
are inappropriate, uneconomic or unachievable (Reznik
1996).

Different fungal species act as a promising source for
the production of various compounds that can be used as
potential herbicides. Since many of these toxins play a key
role in the development of plant diseases (Pointing and
Hyde 2001); the potential of these chemicals as herbicides
can also be explored. When it comes to controlling weeds,

herbicide-resistant weeds can be a challenge for conven-
tional control methods. Therefore, there is a potential to
find compounds that can act as models for developing
herbicides with new modes of action (Castro de Souza
et al. 2016). Through in depth studies on the potential of
fungi and their products, more environmentally friendly
herbicides can be produced for sustainable and eco-friendly
control of weeds.

15. Fungal antagonists used in post-harvest
disease control

There are a myriad of post-harvest applications for fungal
antagonistic agents however there are several challenges
during the commercialization of these biocontrol strains. At
present only two commercial products available as post-
harvest antagonistic agents with a small market share.
Biosave (based on the bacterium *Pseudomonas syringae*)
has been used in the USA to control diseases of sweet
potato and potato diseases. “Shemer” (based on the yeast
*Metschnikowia fructicola*) has been applied in Israel
commercially to control post-harvest rots of sweet potato
and carrot (Droby et al. 2009). Large-scale feasibility tests
are warranted before the antagonistic strains are to be
applied to fresh commodities. For the successful imple-
mentation of biocontrol strategies, the combination of
commercial settings, industrial support, and quality control
mechanisms to build up the confidence of farmers are all
critical factors in the field. Some examples of potential
fungi that can be used in post-harvest applications are
provided below.

Saccharomycetes: *Candida* is a genus that comprises some commonly used antagonistic yeasts that can reduce post-harvest decay of several fresh commodities. For example, *Candida guilliermondii* has been used to control gray mold caused by *Botrytis cinerea* in nectarines and peach (Tian et al. 2002) and *C. communis* is effective against *Aspergillus carbonarius* and *A. niger* (which produce ochratoxin A) in grape berries (Bleve et al. 2006). The volatile organic compounds of *C. intermedia* reduce *Botrytis* fruit rot in strawberries (Huang et al. 2011). *Candida oleophila* has controlled banana crown rot caused by *Colletotrichum musae*, *Fusarium moniliforme* and fungal complexes (Lassois et al. 2008). Droby et al. (2009) applied *Candida saitoana* solely against *Penicillium digitatum* and *Botrytis cinerea* on the post-harvest rots affecting pome and citrus fruits. *Candida membranifaciens* has reduced post-harvest anthracnose disease caused by *Colletotrichum gloeosporioides* in mangos (Kefialew and Ayalew 2008). In vitro application of *Pichia anomala* (also known as *Wickerhamomyces anomalus*) has controlled post-harvest decay of apples (Santos et al. 2004) and bunch rot of table grapes (Parafati et al. 2015). In addition, the
in vivo application of *Pichia anomala* has reduced crown rot disease in banana (Lassois et al. 2008). *Saccharomyces boulardii* can induce phytoalexin formation on sorghum and soybean (Stangarlin et al. 2010). *Saccharomyces cerevisiae* inhibited the activity of *Botrytis cinerea* (in grapes) by producing volatile compounds (Parafati et al. 2015). In vitro application of *Metschnikowia pulcherrima* against post-harvest rot of grapes (Bleve et al. 2006, Parafati et al. 2015) was also notably successful.

**Other Ascomycota:** *Aureobasidium pullulans* has been used to control bunch rot in table grapes caused by *Botrytis cinerea* (Parafati et al. 2015). In addition, post-harvest rot of table grapes caused by *Monilinia laxa* (Schena et al. 2003) were also controlled successfully by this fungus. *Penicillium frequentans* has been shown to effectively inhibit the growth of *Monilinia* sp., which causes brown rot in peaches (Guijarro et al. 2007).

**Basidiomycota:** *Cryptococcus magnus* can inhibit the mycelial growth of *Colletotrichum gloeosporioides* in vitro, and controlled post-harvest anthracnose in papaya (de Capdeville et al. 2007). An aqueous extracts from the basidiomes of *Lentinula edodes* has controlled the growth of *Puccinia recondita* f. sp. *tritici* (Fiori-Tutida et al. 2007). In addition, the in vivo application of *Pycnoporus san-guineus* has controlled the angular leaf spot in beans caused by *Pseudocercospora griseola* (Viecelli et al. 2009). Aqueous extracts of the basidiomes of *Agaricus sub-rufescens* have also shown an antagonism against *Puccinia recondita* f. sp. *tritici*. (Fiori-Tutida et al. 2007). However, these basidiomycetes and their extracts have not been proven to be effective in field trials, and the development of methods for effective mass production to attain favor-able costs of goods will constitute a serious problem.

## 16. Biocontrol of rusts and smuts by antagonistic fungi

Rust fungi (Uredinales) are one of the largest groups in the Basidiomycota, comprising about 5000–6000 species found on a wide range of hosts, including ferns, gymnosperms, and mono- and dicotyledonous angiosperms (Alexopoulos et al. 1996). Diseases such as coffee leaf rust, *Hemileia vastatrix*, wheat stem rust, *Puccinia graminis*, *Melampsora* leaf rusts of Salicaceae (*Populus* and *Salix*) and *Cronartium* stem rusts of hard pines are causing enormous losses and often making it necessary to replace susceptible crops entirely with non-host species (Littlefield 1981).

Smuts primarily affect grasses viz corn (maize), wheat, sugarcane, barley, oats, forage grasses and sorghum (Feldbrügge et al. 2013). A smut is characterized by spores that accumulate in soot-like masses called sori, which are

formed within blisters in seeds, leaves, stems, flower parts, and bulbs (Laurie et al. 2012). The sori usually break up into a black powder that is readily dispersed by the wind. Many smut fungi enter embryos or seedling plants, then develop systemically, and appear externally only when the plants are nearing maturity (Liu et al. 2017a). Currently, the most widely used control method for sugarcane smut disease is the breeding of resistant cultivars (Shen 2002; Wada 2003; Croft et al. 2008; Lwin et al. 2012; Shen et al. 2014). However, its development is constrained by long breeding processes, high costs, and the availability of smut-resistant parental lines. Disease attributed to smut fungi could also be controlled by soaking seed canes with chemical fungicides (Olufolaji 1993; Bhuiyan et al. 2012). Another approach is using plant or fungal extracts that inhibit smut pathogen germination and growth (Lal et al. 2009). A large number of fungi have been identified as hyperparasites of rust and smut fungi, which are being used as biocontrol agents worldwide (Gowdu and Balasubramanian 1988; Kranz 1981; Feldbrügge et al. 2013).

Various studies support the ability of certain fungi to control the growth of smuts and rusts. The mechanisms through which biocontrol agents act are antibiosis, secretion of metabolites that are toxic, lytic enzymes, parasitism and competition for nutrients. Figure 3 shows the different mechanisms of antagonistic fungal species action. Biological approaches are gaining popularity, including the use of microbial antagonists (Eckert and Ogawa 1988). *Cladosporium* species co-exist with rust sori, and some are believed to be invariably hyperparasites of Uredinales (Moricca et al. 1999). *Cladosporium uredinicola* is a common necrotrophic hyperparasite that can destroy rust hyphae and causes coagulation and disintegration of the cell cytoplasm of *Puccinia cestri* (Spegazzini 1912), *Puccinia* (Ellis 1976), *Cronartium quercuum* (Morgan-Jones and McKemy 1990), *Puccinia violae* (Traquair et al. 1984) and *Puccinia horiana* (Srivastava et al. 1985). Moreover, *C. uredinophilum* was also reported to colonize and destroy *Uredo cyclotrauma* propagules in Paraguay (Spegazzini 1912). Steyaert (1930) also described *C. hemileiae* as an effective hyperparasite of coffee rust fungus, *Hemileia vastatrix*, in Zaire (Democratic Republic of Congo). Powell (1971) reported that *C. gallicola* in galls of *Cronartium comandrae* on *Pinus contorta* var. *latifolia* is parasitic on aeciospores. Hyphae of *C. gallicola* penetrate into the aeciospores of pine gall rust, *Endocronartium harknessii* (Sutton 1973). Tsuneda and Hiratsuka (1979) investigated *C. gallicola* and found that it parasitized *E. harknessii* by both simple contact—disintegrating the cell walls of the spores—and by actual penetration of the spore walls, with or without the formation of appressoria, causing the coagulation and disappearance of the host cytoplasm.
Hulea (1939) and Rayss (1943) documented a similar phenomenon where *C. aecidiicola*, a common hyperparasite of rusts in Europe and in the Mediterranean area, parasitized *E. harknessii* on *Pinus* spp. in California (Byler et al. 1972). Keener (1954) stated that this hyperparasite also drastically parasitized aecia of *Puccinia conspicua* in Arizona and urediniospores of *Melampsora medusae* under storage conditions (Sharma and Heather 1980). Moreover, Srivastava et al. (1985) also documented that *Puccinia horiana* was often regulated by *Cladosporium sphaerospermum* and *C. tenuissimum*. They were also detected from aeciospores of the two-needle pine stem rust *Cronartium flaccidum* (Moricca et al. 1999). Other groups of fungi aside from *Cladosporium* were also reported to act as biocontrol agents. The entomopathogenic and mycopathogenic fungus *Lecanicillium lecanii* is also known to attack coffee leaf rust, *Hemileia vastatrix* (Jackson et al. 1997).

Not many commercial biofungicides for rust and smuts based on antagonistic fungi are currently available. Mahmud and Hossain (2016) showed that the BAU-biofungicide (2%) (*Trichoderma* based preparation) significantly affected the mycelial growth of *Ustilaginoidea virens* in an in-vitro test, but this observation remains to be confirmed in greenhouse and field trials.

Kranz (1981) documented more than 80 species of fungi from over 50 genera reported as hyperparasites of rusts. However, this number might be an overestimate due to some taxa being synonyms. Even though this large number of antagonistic fungi on rusts and smuts has been reported, few commercially, improved biofungicides are available for practical application. As in other applications of biocontrol agents (see the above chapters), product formulation is the most critical step of the entire development process (Janisiewicz and Jeffers 1997). The next few years will likely see the increased application of biocontrol agents in agriculture, with particular emphasis on the use of mixtures of antagonists on the same plant organ. This approach may lead to a wider spectrum of activity of the biological treatment or an increase in either the efficacy or consistency of the biological treatment. Furthermore, collaborative work of academic, federal and private sector scientists is necessary to develop more effective and consistent biofungicides.

## Enhancing crops and forestry

In this section, we report on the ways in which fungi are being used or may be used in the future in enhancing plant development within agriculture, forestry and horticulture.

## 17. Biofertilizers

Biofertilizers are produced from organic matter or agro-industrial wastes, which act as substrate for propagation of inoculum of selected microorganisms (Kaewchai et al. 2009). There are two approaches to developing potential biofertilizers: either the application of a single superior species with multifunctions, or groups of microorganisms (consortia) beneficial to plants (Vassilev et al. 2015). Biofertilizers have been used in agriculture, horticulture, landscape restoration, and soil remediation since the late 1980s (Hart and Trevors 2005). The long-term use of biofertilizers is economical and also eco-friendly to plant, animal and human health, and biofertilizers are renewable and low-cost resources which are accessible to marginal and small farmers (Dubey and Maheshwari 2008; Pal et al. 2015). Thus, the use of biofertilizers is recommended over chemical fertilizers. Details regarding biofertilizers such as term, role, types and advantages have been described by Kaewchai et al. (2009), Pal et al. (2015), Vassilev et al. (2015) and Itelima et al. (2018).

Several studies have applied fungal inocula as biofertilizers in greenhouse and/or field trials (Grigera et al. 2007; Rahi et al. 2009; Goetten et al. 2016; Zhang et al. 2016a; Wang et al. 2018c). Mycorrhizal fungi are widely used in agriculture, as they form root symbiotic relationships and provide many benefits to plants, such as improved plant growth and development, increased nutrient uptake and enhanced plant tolerance to disease (Whipps 2004; Liu and Chen 2007; El-Shaikh and Mohammed 2009; Smith et al. 2010; Hernández-Montiel et al. 2013; Goetten et al. 2016; Janoušková et al. 2017). Strains of the genera, such as *Alternaria*, *Aspergillus*, *Chaetomium*, *Fusarium*, *Penicillium*, *Serendipita* (*Piriformospora*), *Phoma*, and *Trichoderma* have been reported as plant growth promoting fungi (Soytong et al. 2001; Muhammad et al. 2009; Salas-Marina et al. 2011; Varma et al. 2012; Bitas et al. 2015; Murali and Amruthesh 2015; Zhang et al. 2016a; Zhou et al. 2018). Examples of the use of fungal inocula treatments on plants are provided in Table 8. These potential plant growth-promoting fungi can be further researched and developed as potent fungal biofertilizers.

Numerous commercial fungal biofertilizer products have been manufactured globally and are available on the market today. There are various formulation types, such as granules, wettable powder, pellets and liquids, which comprise one or multiple fungal inocula. *Aspergillus*, *Chaetomium*, *Penicillium* and *Trichoderma* species have been used in biofertilizer products. For example, Ketomium® has been developed and improved from strains of *Chaetomium* spp. in pellet and powder form. The product was used in greenhouse and field trials of tomato,
<table><caption>**Table 8** Examples for the use of fungal (and oomycete) inocula treated on plants</caption><thead><tr><th>Fungal inoculum</th><th>Treated plant</th><th>Result</th><th>References</th></tr></thead><tbody><tr><td>Arbuscular mycorrhiza</td><td>Zea mays</td><td>Active during the reproductive growth stages and may benefit high productivity of maize crops by facilitating P uptake</td><td>Grigera et al. (2007)</td></tr><tr><td>Arbuscular mycorrhiza</td><td>Citrullus lanatus</td><td>Reduced replant problems through effective modification of the soil microbot structure, and by increasing the soil enzyme activities</td><td>Zhao et al. (2010)</td></tr><tr><td>Arbuscular mycorrhiza (Rhizophagus clarus and Claroideoglomus etunicatum)</td><td>Woody plant seedlings of various plants</td><td>- Increased root colonization of all woody plant seedlings<br>- Increased plant height and stem diameter of L. divaricata, C. robustum and C. fissilis<br>- Increased shoot biomass growth of L. divaricata, C. robustum, G. gardneriana and C. fissilis<br>- Increased shoot phosphorus of C. robustum, S. terebinthifolius and G. gardneriana</td><td>Goetten et al. (2016)</td></tr><tr><td>Aspergillus niger (CS-1)</td><td>Wheat</td><td>Promoted plant growth by increasing the fresh and dry mass of wheat per plant in pot experiments</td><td>Wang et al. (2018c)</td></tr><tr><td>Discosia sp. (HKUCC 6626)</td><td>Zea mays, Pisum sativum, Cicer arietinum</td><td>Increased root length, shoot length and dry matter in the test plants over the uninoculated control under the controlled environment</td><td>Rahi et al. (2009)</td></tr><tr><td>Endophytic strains of Fusarium tricinctum (RSF-4L) and Alternaria alternata (RSF-6L)</td><td>Rice plants</td><td>Enhanced growth attributes, including increased root-shoot lengths, chlorophyll contents, and biomass</td><td>Khan et al. (2015a)</td></tr><tr><td>Penicillium sp., Trichoderma sp., (Pythium sp. (Oomycete))</td><td>Pearl millet seeds</td><td>- Penicillium sp. at 5% (w/w) concentration recorded highest seed germination of 92% and 1701.9 seedling vigor<br>- Penicillium sp. at 5% (w/w) and Pythium sp. at 10% (w/w) showed maximum disease protection of 67% and 61% respectively against downy mildew disease of pearl millet<br>- Penicillium sp. and Trichoderma sp. recorded highest disease protection of 71% and 66%, respectively under greenhouse conditions</td><td>Murali et al. (2012)</td></tr><tr><td>Purpureocillium lilacinum</td><td>Tomato seeds</td><td>Increased of the percentage of tomato seed germination from 71 to 85% after 48 h</td><td>Cavello et al. (2015)</td></tr><tr><td>Trichoderma longibrachiatum</td><td>Wheat seeds</td><td>Increased wheat seedling height and root length, compared to the NaCl stress treatment</td><td>Zhang et al. (2016a)</td></tr></tbody></table>

corn, rice, pepper, citrus, durian, bird of paradise and carnation plants in Thailand (Soytong et al. 2001). Plants treated with Ketonium® showed better plant growth and higher yield than non-treated control plants. In addition, Ketonium® had the ability to control *Phytophthora* sp., causing citrus root rot in the field. Other examples of fungal biofertilizer products are given in Table 8.

Biofertilizers increase the uptake of nutrients from the soil or atmosphere, and produce bioactive compounds, enzymes and hormones which stimulate plant growth and enhance root growth (Chi et al. 2010; Abdel-Fattah et al. 2013; Pal et al. 2015). Fungal biofertilizers are able to solubilize and mobilize unavailable organic and inorganic

forms of phosphorus into soluble forms, making them available to plants. For example, *Aspergillus niger* was mixed with *Bacillus megaterium* to form phosphate solubilizing microorganisms. These microorganisms were applied as biofertilizers in India (Pal et al. 2015). Arbuscular mycorrhizae have been used as phosphate mobilizing biofertilizers (Zhang et al. 2018). Biofertilizers play an important role in the recycling of plant nutrients and in enhancing the rate of compost degradation (Pal et al. 2015). Some biofertilizers act as antagonists and suppress the incidence of soil borne plant pathogens while helping in the biocontrol of plant diseases (Thamer et al. 2011; Pal et al. 2015).
Fungal derived stimulants, or elicitors, are fungi or fungal compounds that enhance the production of secondary metabolites, or elicit growth or immune response in a target plant species upon application. Plant responses include the upregulation of genes involved in plant defense, as well as the increased production of antimicrobial compounds, lignin, secondary metabolites, and certain proteins (Vassilev et al. 2015). Potential uses for such elicitors include the enhanced production of commercially valuable compounds/metabolites, or the artificial enhancement of plant defenses when pathogens are detected (Radman et al. 2003). A novel approach for the use of elicitors is to incorporate them with immobilized stimulants, such as with arbuscular mycorrhizal inoculum. Additionally, plant-derived elicitors, which enhance the growth and development of beneficial fungi such as arbuscular mycorrhizae, also show promise in advancing this field of study (Akiyama et al. 2005; Besserer et al. 2006). Elicitors have a high potential for enhancing plant productivity and improving plant defenses against pathogens, and given that elicitors can be used in combination with other types of biofertilizers, they hold much potential for wide scale application in the future.

Fungal biofertilizers are applied on a very small scale in agriculture as compared to chemical fertilizers due to their limited shelf life and slower rate of effect. Olivian et al. (2004) reported using sterilized peat as solid support for *Fusarium oxysporum* inoculation, storing this admixture at room temperature without loss of activity. Growth and formulations based on recycling agro-industrial wastes can be expected to employ nitrogen-fixing and other microorganisms with different characteristics, such as biocontrol, P-solubilization, lignocellulolytic activity. For example, combinations between *Trichoderma* spp. and P-solubilizing fungi can be cultured based on agroindustrial-wastes, leading to mineralization of the matrix/substrate by the combined enzyme actions. We could apply immobilization of fungal cells together with enhanced biotechnology and in combination with elicitors. Immobilized cell technologies permit the use of two and more microorganisms, which result in highly effective synergies benefiting all the organisms involved, including the plants (Vassilev et al. 2015). In order to effectively implement the use and gain the full benefits of biofertilizers, an integrated approach engaging a variety of mechanisms should be considered. Such an approach could be tailored to suit specific industry needs and target defined outcomes, such as improved growth, upregulation of key metabolites, or enhanced plant defenses.

## 18. Arbuscular mycorrhizae as biofertilizers

Ectomycorrhizal association describes a structure that results from a mutualistic symbiosis between the roots of higher plants and root-inhabiting fungi. Within this symbiotic relationship, the role of the fungi is to help the host plants take up water and nutrients, receiving plant-derived carbohydrates from photosynthesis in return. About 6000 plant species in 145 genera and 26 families (approximately 5600 angiosperms and 285 gymnosperms) have been estimated to possess ectomycorrhizal symbiotic fungal partners (Brundrett 2009; Tedersoo et al. 2010). Ectomycorrhizal association helps both the fungi and their host plants to overcome environmental stresses caused by low nutrients, drought, disease, extreme temperatures and heavy metal contamination (Smith and Read 2008; Courty et al. 2010; Kipfer et al. 2012; Heilmann-Clausen et al. 2014). Moreover, ectomycorrhizae can improve soil structure and nutrients; protect the plants against root pathogens; promote plant growth by producing phytohormones; and increase the photosynthetic rate of the plants (Splivallo et al. 2009; Ramachela and Theron 2010; Makita et al. 2012). Ectomycorrhizae are dominated by members of the Basidiomycota, some Ascomycota, and, rarely, Mucoromycota (Taylor and Alexander 2005; Rinaldi et al. 2008; Tedersoo et al. 2010). Generally, ectomycorrhizae produce reproductive fruiting bodies appearing above- or below-ground that are essential to the food webs of forest ecosystems and their spore dispersal (Rinaldi et al. 2008; Wilson et al. 2011).

Plant seedling regeneration and restoration are of pivotal interest to forestry, but the survival of seedlings is often poor both in nurseries and natural plantation areas, especially in mine spoils, polluted areas, and other treeless areas. Therefore, the main purpose for the application of ectomycorrhizae is to improve the survival and growth of seedlings. The potential advantages of ectomycorrhizal association in nurseries are not only the positive growth responses of the seedlings, but also a reduction of fertilization costs in an environmentally friendly manner. The role of ectomycorrhizae in forest establishment and recovery has been well-established. Numerous studies on the ectomycorrhizae inoculation of seedlings have shown increases in plant growth and productivity, the viability of seedlings, and seedling establishment on a forest restoration programs (Teste et al. 2009; Dalong et al. 2011; Brearley et al. 2016; Velmala et al. 2018). Ectomycorrhizae are particularly important for the growth of economically important trees, including species of beech (*Fagus*), dipterocarps (*Dipterocarpus* and *Shorea*), eucalyptus (*Eucalyptus*), oak (*Quercus* and *Castanopsis*), pine
(Pinus) and spruce (Picea) (Tennakoon et al. 2005; Flykt et al. 2008; Dalong et al. 2011; Kayama and Yamanaka 2016). *Cenocococum, Pisolithus, Laccaria, Rhizopogon, Russula, Scleroderma* and *Thelephora* species have been shown to increase the rate of survival and growth of eucalyptus, pine and oak seedlings in both plantation and reforestation programs (Fig. 16) (Chen et al. 2006; Jha et al. 2008; Cram and Dumroese 2012; Kipfer et al. 2012; Zong et al. 2015).

Generally, three main types of ectomycorrhizal inoculants—soil, fruiting body/spore and vegetative mycelium—have been used in nurseries. Forest soil was used as a source of indigenous ectomycorrhizal fungi in an inoculation experiment mixed with planting substrates (Kaewgrajang et al. 2013; Dulmer et al. 2014; Restrep-Liano et al. 2014; Livne-Luzon et al. 2017). This method is still used in many parts of the world, particularly in developing countries. However, the use of forest soil inoculants has the major disadvantage that the ectomycorrhizal composition is unknown. Moreover, it requires large amounts of soil and hence risks introducing plant pathogens and weeds exits. Fruiting bodies/spores of various ectomycorrhizae are easily obtained from natural forests and can be easily applied to plant seedlings as inoculants. The variety of application methods include mixing with sand, clay, or vermiculite carrier before being added to planting substrate or soil, suspension in water and drenching or irrigating, spraying, and

encapsulation or coating onto seeds. Ectomycorrhizae that are “gasteromycetes” (puffball fungi) with conspicuous basidomes are better sources than the gilled fungi if large numbers of spores are required, as they are easier to collect and use. For instance, species of the genera *Pisolithus*, *Rhizopogon* and *Scleroderma* produce a large quantity of spores, and the approximate spore concentration in a seedling inoculation may range from $10^5–10^7$ spores/ml (Chen et al. 2006; Bruns et al. 2009; Rai and Varma 2011; Aggangan et al. 2013). Most previous studies resulted in acceptable levels of ectomycorrhizal association, improved seedling growth of pines in the nursery, and improved outplanting success following inoculation with *Pisolithus* and *Rhizopogon* spores (Bruns et al. 2009; Dalong et al. 2011).

There are of course limitations to fruiting body/spore inoculants: only those ectomycorrhizal species able to produce large numbers of fruiting bodies and spores can be used, and there may be a concern about the compatibility and efficiency of ectomycorrhizae to the plant species to be cultivated. As an alternative, vegetative mycelial inoculants obtained from pure cultures of ectomycorrhizae may be prepared from a pure culture using different methods, e. g. using mycelial suspensions and substrate carriers such as forest litter, cereal grains, peat moss, vermiculite, and alginate-beads (de Oliveira et al. 2006; Rossi et al. 2007; Lee et al. 2008a, b; Restrep-Liano et al. 2014; Kayama and Yamanaka 2016; Kumla et al. 2016). This inoculant type

<figure><img src="image_26.png" /><figcaption>**Fig. 16** Arbuscular mycorrhizae inoculum production. **a** Pot culture of sorghum and maize, **b** on-farm inoculum production using leaf litter compost and agricultural wastes; **c** In vitro production with root organ culture; **d** newly produced *Funneliformis mosseae* spores attached to Ri T-DNA transformed carrot roots</figcaption></figure>
has proven to be the most suitable method because of their efficiency in promoting plant growth by selected fungal isolates. However, optimal conditions, including nutrition, temperature and substrate carrier, must always be established empirically for large-scale production.

Several commercial ectomycorrhizal products have been developed. For instance, the commercial mycelial inoculants of MycoRhiz®, Ectomycorrhiza Spawn®, Somycel PV and Mycobead® are available. BioGrow Blend®, MycoApply®-Ecto, Ectovit® and Mycor Tree® Ecto-Injectable are commercially available products with ectomycorrhizae spores. The commercial products produced by mixing endomycorrhizae and ectomycorrhizae spores are MycoApply®-Endo/Ecto, BioOrganic™ Mycorrhizal Landscape Inoculant and Mycoke® Pro ARBOR·WP. In order to apply ectomycorrhizae in forestry, it is necessary to select ectomycorrhizal isolates of high compatibility and efficiency in the colonization of the target plant species. Inoculant types, as well as inoculation protocols and skills in nursery practices, lead to the success of an inoculation program under the proper environmental conditions in the plantation site.

The potential for arbuscular mycorrhizae to increase
crop yields has been known for decades, but there are few
published studies demonstrating the effectiveness of the
large-scale inoculation of globally important crops, espe-
cially in the tropics where population growth is high (Ro-
driguez and Sanders 2015). Therefore, researchers need to
study large-scale arbuscular mycorrhizae application to
crops in the tropics where phosphate bioavailability is low
and the application of arbuscular mycorrhizae has the
strongest potential to increase food production and reduce
the need to apply phosphate fertilizers (Ceballos et al.
2013). Manufacturers should ensure their arbuscular myc-
orrhizae products are free from other microorganisms and
ensure product quality and sufficient weight for cheap
transport. Farmers should have easy access to arbuscular
mycorrhizae products, correctly apply them to the crops,
and know how to produce on-farm arbuscular mycorrhizae
inoculum for sustainable agriculture.

19. Application of ectomycorrhizal fungi
in forestry

Ectomycorrhizal association describes a structure that
results from a mutualistic symbiosis between the roots of
higher plants and root-inhabiting fungi. Within this sym-
biotic relationship, the role of the fungi is to help the host
plants take up water and nutrients, receiving plant-derived
carbohydrates from photosynthesis in return. About 6000
plant species in 145 genera and 26 families (approximately
5600 angiosperms and 285 gymnosperms) have been esti-
mated to possess ectomycorrhizal symbiotic fungal

partners (Brundrett 2009; Tedersoo et al. 2010). Ectomy-
corrhizal association helps both the fungi and their host
plants to overcome environmental stresses caused by low
nutrients, drought, disease, extreme temperatures and
heavy metal contamination (Smith and Read 2008; Courty
et al. 2010; Kipfer et al. 2012; Heilmann-Clausen et al.
2014). Moreover, ectomycorrhizae can improve soil
structure and nutrients; protect the plants against root
pathogens; promote plant growth by producing phytohor-
mones; and increase the photosynthetic rate of the plants
(Splivallo et al. 2009; Ramachela and Theron 2010; Makita
et al. 2012). Ectomycorrhizae are dominated by members
of the Basidiomycota, some Ascomycota, and, rarely,
Mucoromycota (Taylor and Alexander 2005; Rinaldi et al.
2008; Tedersoo et al. 2010). Generally, ectomycorrhizae
produce reproductive fruiting bodies appearing above- or
below-ground that are essential to the food webs of forest
ecosystems and their spore dispersal (Rinaldi et al. 2008;
Wilson et al. 2011).

Plant seedling regeneration and restoration are of pivotal
interest to forestry, but the survival of seedlings is often
poor both in nurseries and natural plantation areas, espe-
cially in mine spoils, polluted areas, and other treeless
areas. Therefore, the main purpose for the application of
ectomycorrhizae is to improve the survival and growth of
seedlings. The potential advantages of ectomycorrhizal
association in nurseries are not only the positive growth
responses of the seedlings, but also a reduction of fertil-
ization costs in an environmentally friendly manner. The
role of ectomycorrhizae in forest establishment and
recovery has been well-established. Numerous studies on
the ectomycorrhizae inoculation of seedlings have shown
increases in plant growth and productivity, the viability of
seedlings, and seedling establishment on a forest restora-
tion programs (Teste et al. 2009; Dalong et al. 2011;
Brearley et al. 2016; Velmala et al. 2018). Ectomycor-
rizae are particularly important for the growth of eco-
nomically important trees, including species of beech
(Fagus), dipterocarps (Dipterocarpus and Shorea), euca-
lyptus (Eucalyptus), oak (Quercus and Castanopsis), pine
(Pinus) and spruce (Picea) (Tennakoon et al. 2005; Flykt
et al. 2008; Dalong et al. 2011; Kayama and Yamanaka
2016). Cenocococum, Pisolithus, Laccaria, Rhizopogon,
Russula, Scleroderma and Thelephora species have been
shown to increase the rate of survival and growth of
eucalyptus, pine and oak seedlings in both plantation and
reforestation programs (Fig. 17) (Chen et al. 2006; Jha
et al. 2008; Cram and Dumroese 2012; Kipfer et al. 2012;
Zong et al. 2015).

Generally, three main types of ectomycorrhizal inocu-
lants—soil, fruiting body/spore and vegetative mycelium—
have been used in nurseries (Fig. 18a). Forest soil was used
as a source of indigenous ectomycorrhizal fungi in an
<figure><img src="image_27.png" /><figcaption>**Fig. 17** Application of *Pisolithus albus* in eucalyptus (*Eucalyptus camaldulensis*) seedlings after 3 months of inoculation (a). T1 *Pisolithus* inoculation experiment. T2 Nutrient solution experiment. T3 Uninoculated experiment. Ectomycorrhizal root tip of *Pisolithus albus* (b). Cross section of ectomycorrhizal root tip of *E. camaldulensis* showed mantle sheath (M) and Hartig net (arrowed). Scale bar B = 1 mm, C = 20 µm</figcaption></figure>

<figure><img src="image_28.png" /><figcaption>**Fig. 18** Inoculant types of ectomycorrhizae (*Pisolithus orientalis*). **a** Soil inoculant, **b** spore suspension inoculant, **c**, **d** vegetative mycelial inoculants</figcaption></figure>

inoculation experiment mixed with planting substrates (Kaewgrajang et al. 2013; Dulmer et al. 2014; Restrep-Liano et al. 2014; Livne-Luzon et al. 2017). This method is still used in many parts of the world, particularly in developing countries. However, the use of forest soil inoculants has the major disadvantage that the

ectomycorrhizal composition is unknown. Moreover, it requires large amounts of soil and hence risks introducing plant pathogens and weeds exits. Fruiting bodies/spores of various ectomycorrhizae are easily obtained from natural forests and can be easily applied to plant seedlings as inoculants. The variety of application methods include mixing with sand, clay, or vermiculite carrier before being added to planting substrate or soil, suspension in water and drenching or irrigating, spraying, and encapsulation or coating onto seeds. Ectomycorrhizae that are “gasteromycetes” (puffball fungi) with conspicuous basidomes are better sources than the gilled fungi if large numbers of spores are required, as they are easier to collect and use. For instance, species of the genera *Pisolithus*, *Rhizopogon* and *Scleroderma* produce a large quantity of spores, and the approximate spore concentration in a seedling inoculation may range from $10^5–10^7$ spores/ml (Chen et al. 2006; Bruns et al. 2009; Rai and Varma 2011; Aggangan et al. 2013). Most previous studies resulted in acceptable levels of ectomycorrhizal association, improved seedling growth of pines in the nursery, and improved outplanting success following inoculation with *Pisolithus* and *Rhizopogon* spores (Bruns et al. 2009; Dalong et al. 2011).

There are of course limitations to fruiting body/spore inoculants: only those ectomycorrhizal species able to produce large numbers of fruiting bodies and spores can be used, and there may be a concern about the compatibility and efficiency of ectomycorrhizae to the plant species to be cultivated. As an alternative, vegetative mycelial inoculants obtained from pure cultures of ectomycorrhizae may be prepared from a pure culture using different methods, e.
g. using mycelial suspensions and substrate carriers such as forest litter, cereal grains, peat moss, vermiculite, and alginate-beads (de Oliveira et al. 2006; Rossi et al. 2007; Lee et al. 2008a, b; Restrep-Liano et al. 2014; Kayama and Yamanaka 2016; Kumla et al. 2016). This inoculant type has proven to be the most suitable method because of their efficiency in promoting plant growth by selected fungal isolates. However, optimal conditions, including nutrition, temperature and substrate carrier, must always be established empirically for large-scale production.

Several commercial ectomycorrhizal products have been developed In order to apply ectomycorrhizae in forestry, it is necessary to select ectomycorrhizal isolates of high compatibility and efficiency in the colonization of the target plant species. Inoculant types, as well as inoculation protocols and skills in nursery practices, lead to the success of an inoculation program under the proper environmental conditions in the plantation site.

## 20. Use of orchid mycorrhizae and endophytes in biotechnology

Orchidaceae is one of the largest families of flowering plants with over 700 genera and 25,000 species (Dearnaley 2007; Sathiyadash et al. 2012). Orchids are found in a wide range of habitats and may grow autotrophically or heterotrophically (Sathiyadash et al. 2012; Tan et al. 2014; Fochi et al. 2017). Orchids are economically very important and their sales represent around 8% of the world floriculture trade. The economically most important genera are *Cymbidium*, *Dendrobium*, and *Phalaenopsis* (Dearnaley 2007, 2016; Chugh et al. 2009; Emsa-art et al. 2018). Some orchids, such as *Gastrodia* (Griesbach 2002; Dearnaley 2007), *Dendrobium officinale* and *D. nobile* are used as natural medicines (Li et al. 2009). Furthermore, the economically most important orchid products are the flavours derived from some species of the genus *Vanilla*, which are grown at a large scale and used in food and drinks (Dearnaley 2007; Gonzalez-Chavez et al. 2018).

Most orchids rely on mycorrhizal fungi for survival, as they are essential for seed germination and early plant growth (Sathiyadash et al. 2012; Fochi et al. 2017). Different fungal symbiotic mycorrhizae have been recorded from orchids (Table 9). Orchid associated non-mycorrhizal endophytic fungi have been investigated via healthy plant organs including leaves, roots and stems (Ma et al. 2015a), whereas mycorrhizal fungi are generally isolated from root tissues (Tan et al. 2014; Ma et al. 2015a, b). Non-mycorrhizal endophytic fungi represent over 110 genera, including Sordariomycetes (*Neonectria*, *Trichoderma*, *Nigrospora*, *Pestalotiopsis*) and Dothideomycetes (*Cercospora*, *Lasiodiplodia*, *Phyllosticta*) (Ma et al. 2015a, b). Dark septate endophytes isolated from *Dendrobium* and *Leptodontidium* sp. enhanced seedling development of *Dendrobium nobile* by forming peloton-like structures in the cortical cells of the orchid (Hou and Guo 2009; Ma et al. 2015a, b). *Fusarium* species promoted seed germination of *Cypripedium* and *Platanthera* orchids (Ma et al. 2015a, b). The endophyte *Umbelopsis nana* isolated from *Cymbidium* spp., promoted growth of *Cymbidium hybridum* (Ma et al. 2015a, b).

Many epiphytic and terrestrial orchids produce minute seeds with minimal nutrient reserves, and lack nutrients for seed germination and development in the early growth stage (Cameron et al. 2006, Sathiyadash et al. 2012; Tan et al. 2014). After germination, orchid seeds produce a protocorm (a preseedling stage/ early stage of the plant) that lacks chlorophyll (Leake 2004; Sathiyadash et al. 2012; Fochi et al. 2017). Protocorms grow in complete dependence on fungal symbionts for nutrients and organic carbon supply (Cameron et al. 2006; Dearnaley 2007). Orchid seedlings develop photosynthetic leaves later and then mature roots are colonized by mycorrhizal fungi (Cameron et al. 2006; Smith and Read 2008; Fochi et al. 2017). The protocorm and mature roots cells are colonized by intracellular fungal coils (pelotons) (Fig. 19) (Dearnaley 2007; Dearnaley et al. 2016; Fochi et al. 2017).

Orchid mycorrhizal associations are useful in the floriculture trade, as they stimulate seed germination and

<table><caption>**Table 9** Fungal symbionts associated with different orchid species</caption><thead><tr><th>Fungal symbionts (Basidiomycota)</th><th>Orchid species</th><th>Locality</th><th>References</th></tr></thead><tbody><tr><td>Rhizoctonia</td><td>Gastrochilus acaulis, Nervilia prainiana and Polystchya concreta</td><td>India</td><td>Senthilkumar (2003) Sathiyadash et al. (2012)</td></tr><tr><td>Ceratobasidium</td><td>Zeuxine strateumatica</td><td>India</td><td>Kumar and Kaushik (2004)</td></tr><tr><td rowspan="3">Tulasnella</td><td>Amerorchis rotundifolia</td><td rowspan="3">Worldwide</td><td>Zelmer et al. (1996)</td></tr><tr><td>Dactylorhiza majalis</td><td>Kristiansen et al. (2001)</td></tr><tr><td>Neuwiedia veratrifolia</td><td>Kristiansen et al. (2004)</td></tr></tbody></table>
<figure><img src="image_29.png" /><figcaption>**Fig. 20** Scheme illustrating the methodology of orchid seed propagation using mycorrhizal fungi (in-vitro conditions) (Nontachaiyapoom et al. 2010; Tan et al. 2014). **a** Effect of mycorrhizal fungi on germination of orchid seeds. **b** Effect of mycorrhizal fungi on growth of orchid seedlings</figcaption></figure>

<figure><img src="image_30.png" /></figure>

propagate orchids (Tan et al. 2014). A brief methodology for the inoculation of mycorrhizal fungus (*Tulasnella* sp.) to orchids according to Nontachaiyapoom et al. (2010) and Tan et al. (2014) is represented in a flow chart (Fig. 20).

*Tulasnella* sp. isolated from roots of *Dendrobium nobile* facilitates significantly higher seed germination of *D. of-ficinale* than that of the control (without inoculation of *Tulasnella* sp.) (Tan et al. 2014). In addition, *Tulasnella* sp.

<figure><img src="image_31.png" /></figure>
<table><caption>**Table 10** Different stages of orchid seed germination (Tan et al. 2014)</caption><thead><tr><td>Stage</td><td>Description</td></tr></thead><tbody><tr><td>0</td><td>No germination, viable embryo</td></tr><tr><td>1</td><td>Enlarged embryo, production of rhizoid(s) (=germination)</td></tr><tr><td>2</td><td>Continued embryo enlargement, rupture of testa, further production of rhizoids</td></tr><tr><td>3</td><td>Appearance of protomeristem</td></tr><tr><td>4</td><td>Emergence of first leaf</td></tr><tr><td>5</td><td>Elongation of first leaf</td></tr></tbody></table>

promotes seed development up to stage 5 (Table 10), while the control without the fungus developed only to stage 2 (Table 10) (Tan et al. 2014). However, fungi isolated from orchid plant roots do not always exhibit functional symbiotic associations with the orchid plant (Dearnaley 2007). Microscopic observations of orchid root sections might be useful to confirm the presence of intracellular fungal mycelium (Nontachaiyapoom et al. 2010; Emsa-art et al. 2018). Furthermore, it is important to evaluate seedling growth of mycorrhizal inoculated orchids under natural conditions (Tan et al. 2014).

Orchid mycorrhizal fungi are also important for controlling disease in floriculture trade (Yoder et al. 2000; Emsa-art et al. 2018). Inoculation of orchid mycorrhizal fungi may enhance plant immunity against pathogenic diseases (Wu et al. 2011; Emsa-art et al. 2018). For example, soft rot disease is one of the most devastating diseases caused by *Dickeya* spp., which kills orchids or causes spots/scars on leaves and flowers (Liau et al. 2003; Emsa-art et al. 2018). One recent study showed that soft rot development in mycorrhizal fungi inoculated orchids was significantly reduced compared to that of non-mycorrhizal fungi inoculated orchids in greenhouse conditions. *Phalaenopsis* is a popular potted plant species that was used for the study by Emsa-art et al. (2018). A brief overview of the methodology of inoculation of mycorrhizal fungi (*Tulasnella deliquescens*) to *Phalaenopsis* to control pathogenic *Dickeya* is presented in Fig. 21.

Several commercial products containing mycorrhizal inoculants exist. These inoculants are available for sale in liquid and powder form for easy and effective usage. Most of these products are organic fertilizers inoculated with mycorrhizae spores and with vitamins, minerals, and nutrients to help bolster the fertility and biological activity of the soil (see Table 11). Mycorrhizal fungi are useful in orchid conservation (Tan et al. 2014). Orchids are a highly diverse plant family and many species may face extinction threats (Reiter et al. 2016) because of habitat loss and overexploitation of attractive species (Dearnaley 2007). With this decline of orchid diversity, it is now an urgent

<figure><img src="image_32.png" /><figcaption>**Fig. 21** Scheme illustrating the methodology for the inoculation of mycorrhizal fungi, *Tulasnella deliquescens* to the orchid *Phalaenopsis* to control pathogenic bacteria (Emsa-art et al. 2018)</figcaption></figure>

requirement to encourage research on the reintroduction of endangered species to natural habitats (Reiter et al. 2016).

## 21. Growth promoting hormones from fungi

Fungi live in diverse habitats and have adapted to ecological niches, including plant systems. Plants and fungi have established complex mutualistic relationships, and wild plants are almost always colonized by endophytic, parasitic and mycorrhizal fungi (Rodriguez et al. 2009; Patkar and Naqvi 2017). Fungi produce a variety of bioactive compounds that play an important role in the physiological activities of the host plant, influencing the growth of the hosts. This can even lead to an increased tolerance to abiotic and biotic stresses of the plants (Pineda et al. 2010). Many studies have shown that fungi enhance plant growth through the solubilization of insoluble minerals in soil and secretion of plant growth regulators (Bilal et al. 2018; Chanclud and Morel 2016; Júnior et al. 2017; Khan et al. 2012). Growth promotion by plant growth regulators or phytohormones production, are signal molecules acting as chemical messengers and play a fundamental role in plants. Plant growth hormones produced by symbiotic fungi may greatly influence processes including
<table><caption>**Table 11** Different products containing mycorrhizal fungi</caption><thead><tr><td>Product</td><td>Mycorrhizae</td><td>Advantages</td><td>Use for</td></tr></thead><tbody><tr><td>Soluble Mycorrhizae<sup>a</sup></td><td>Seven strains of endo-mycorrhizae fungi and five strains ecto-mycorrhizae fungi</td><td>Enhance seed germination and transplants growth and new root development</td><td>Deep root feeding, transplanting plants, shrubs and nurseries</td></tr><tr><td>Soluble root growth enhancer (Oregonism XL)<sup>b</sup></td><td>Six strains of endotrophic and six strains of ectotrophic mycorrhizae species</td><td>Enhance nutrient and water uptake, encourage vigorous root systems and help plants resist stress and disease</td><td>Effective in soil and hydroponics, fruits and flowering plants</td></tr><tr><td>MycoStim (soil inoculant)<sup>a</sup></td><td>Eight strains of mycorrhizae and two species of Trichoderma</td><td>Enhance growth and nutrient uptake, stress resistance, improve plant quality, yields and vigor</td><td>Flowers and vegetables</td></tr><tr><td>Root rally with mycorrhizae<sup>c</sup></td><td>Bio-nutrients and inoculated with mycorrhizae spores</td><td>Improve stress tolerance, encourage root growth and increase water and nutrient absorption</td><td>Trees, shrubs, flowers, fruits and vegetables.</td></tr><tr><td>Natural biological inoculant (soluble powder)<sup>d</sup></td><td>Nine mycorrhizal species, 15 beneficial bacteria and two species of Trichoderma</td><td>Improve nutrient and water uptake for enhanced plant performance</td><td>Flowers and vegetables</td></tr></tbody></table>

seed germination, root initiation, stem and leaf growth, phototropism, flowering and fruit growth (Khan et al. 2015b; Petracek et al. 2003). On the other hand, many fungal pathogens produce phytohormones during host invasion and colonization, which are mainly involved in plant defense response (Kazan and Lyons 2014; Spence et al. 2015).

The use of plant growth hormones in agriculture and horticulture has been growing significantly. One of the best studied plant growth regulators is indole-3-acetic acid (IAA), a principal auxin involved in apical dominance, root initiation, cell division and cell enlargement (Benjamins and Scheres 2008; Vessey 2003). Several fungal strains belonging to various families of Ascomycota, Basidiomycota and Mucoromycota have been reported to produce IAA (Chandra et al. 2018; Hasan 2002; Waqas et al. 2012). Recently, Numponsak et al. (2018) reported that the IAA containing crude extract of a strain of *Colletotrichum fructicola* increased coleoptile elongation of rice, corn and rye, in a similar manner as the commercial IAA standard. Moreover, when the spore suspension of *C. fructicola* was applied in rice seedlings, it accelerated the growth of seedlings and enhanced their biomass and chlorophyll content (Fig. 22).

Gibberellins were first discovered in the culture filtrate of the pathogen “Gibberella” (now classified in *Fusarium*) *fujikuroi*, which causes disease in rice plants (Hedden and Sponsel 2015). This hormone is used to accelerate the processes of seed germination, stem elongation, leaf expansion, flower initiation and fruit development (Yamaguchi 2008). Gibberellic acid can induce bolting and

<figure><img src="image_33.png" /><figcaption>**Fig. 22** Influence of a spore suspension of *Colletotrichum fructicola* CMU-A109 on the growth of rice seedlings after 10 days (right) against the untreated control (left). Bar 2 cm</figcaption></figure>

flowering in rosette species and rescue dwarf mutants of maize and peas (Hedden and Sponsel 2015).

Cytokinins (CKs) were reportedly found in pathogenic fungi such as *Leptosphaeria maculans*, *Magnaporthe oryzae*, and in mycorrhizal fungi (Crafts and Miller 1974; Chanclud et al. 2016; Trdá et al. 2017). This hormone plays a significant role in plant physiological processes including apical dominance, cell division, leaf senescence, chloroplast biogenesis, vascular and shoot differentiation, programmed cell-death and anthocyanin production (Fahad et al. 2014).

Abscisic acid (ABA) is another important plant growth hormone, which was discovered as secondary metabolite of

<footnote><sup>a</sup>https://www.planetnatural.com/product/soluble-mycorrhizae/</footnote>

<footnote><sup>b</sup>https://www.planetnatural.com/product/roots-organics-oregonism-xl/</footnote>

<footnote><sup>c</sup>https://www.planetnatural.com/product/root-rally/</footnote>

<footnote><sup>d</sup>https://www.planetnatural.com/product/myco-madness/</footnote>
various fungi, including species of *Aspergillus*, *Botrytis*, *Cercospora*, *Penicillium* and *Rhizopus* (Shi et al. 2017). Abscisic acid plays a significant role in plant responses and adaptations to various environmental stresses, thus increasing crop yields (Devinar et al. 2013; Narusaka et al. 2003). Moreover, some fungi are able to produce ethylene (ET), salicylic acid (SA) and jasmonic acid (JA) hormones that regulate plant defense against pathogens as well as plant growth and development. *Trichoderma* species have been reported to simultaneously induce the ET, SA and JA pathways following pathogen attacks in *Arabidopsis thaliana*, grape, tomato and melon (Jogaiah et al. 2018).

The production of plant hormones by fungi depends on their growth conditions, such as temperature, pH, incubation period, growth dynamics and internal physiology (Bilal et al. 2018; Khan et al. 2012). Optimization using statistical approach is necessary to improve yield for production of phytohormones and other bioactive metabolites in the industry level (Albermann et al. 2013). Although plant growth regulators are widely found in plants, fungi and bacteria, they are being produced by chemical synthesis at the commercial scale. The high cost and low productivity of the processes available to access microbial-derived plant hormones limit their extensive application and thus restrict the development of the industry (Shi et al. 2017). Currently, ABA is produced with high yielding strains of *B. cinerea* and has been available commercially since 2009 (Rademacher 2015). Furthermore, in case of GAs production, *Fusarium* (*Giberella* fujikuroi produces relatively high titers and therefore it is the principally utilized strain for industrial production. The possibility of the chemical synthesis of GAs was studied, and it was found that the compounds were too complex and the process too expensive to be a commercially viable alternative (Mander 2003). In order to increase the GAs titer, the mutagenesis of *Fusarium fujikuroi* and the use of cell immobilization together with extractive fermentation techniques were successfully applied in the production of GAs (Eleazar et al. 2000; Lale et al. 2006). In general, 60% to 90% of the total applied fertilizer was lost and the remaining 10% to 40% were taken up by plants. Only *Trichoderma* spp. and mycorrhizal fungi are commercially produced and applied in crop production.

Crop production not only faces the challenges of climate change and the diseases that affect them, but also increases in food demands due to the burgeoning global population. Biotic and abiotic stresses are also important limitations on global crop productivity. Hence plant hormones are playing an increasingly significant role in the horticulture and agricultural field. However, the low efficiency of fungal fermentation processes continues to preclude the cost reductions necessary for industrial-scale production. Thus, the study of fungal genome sequencing may help to shed

light on the presence of the hormonal biosynthesis pathways. The molecular biology and transcriptomic analyses of fungal-derived plant hormones may provide more details related to the effects of phytohormones on plants, and ultimately effect the increased productivity of these hormones.

## 22. Mitigating abiotic stress in plants: the endophyte method

Abiotic stresses such as drought, salinity, extreme temperatures and oxidative stresses adversely affect plant growth and productivity (Fahad et al. 2017). Many of these stresses, either individually or in combinations, take a heavy toll on agricultural productivity in most parts of the semi-arid tropics (McCartney and Lefsrud 2018). In recent decades, the effects of such abiotic stresses have been further exacerbated by unprecedented changes in climate (Fedoroff et al. 2010). Conventional crop improvement approaches to render plants tolerant to abiotic stresses and resilient to climate change have been met with limited success, primarily due to the combination of the stressors and the multitude of plant traits involved in determining tolerance. A more recent and exciting approach has emerged from the use of endophytic fungi to alter plant responses and adaptation to abiotic stresses.

Endophytic fungi coexist with plants without causing any apparent disease symptoms. Several studies have demonstrated the role of endophytic fungi in enhancing plant fitness, within both, normal and stressful environments (Abdelaziz et al. 2017). The rationale for an endophyte-based adaptation rests on the fact that the endophytes adapt to environmental adversities faster than their host plants, and often are also able to collaterally share such adaptations with their respective host plants (Suryanarayanan et al. 2017). Careful exploitation of endophytic fungi could offer a strategic approach to alleviate stress effects in non-host plants, such as crop species (Rodriguez et al. 2008). Here, we briefly review studies that have explored the use of endophytes in modulating plant responses to abiotic stresses and discuss how these could potentially be used to mitigate abiotic stressors in crop plants.

Abiotic stresses such as drought, salinity, extreme temperatures and oxidative stresses adversely affect plant growth and productivity (Fahad et al. 2017). Many of these stresses, either individually or in combinations, take a heavy toll on agricultural productivity in most parts of the semi-arid tropics (McCartney and Lefsrud 2018). In recent decades, the effects of such abiotic stresses have been further exacerbated by unprecedented changes in climate (Fedoroff et al. 2010). Conventional crop improvement approaches to render plants tolerant to abiotic stresses and
resilient to climate change have been met with limited success, primarily due to the combination of the stressors and the multitude of plant traits involved in determining tolerance. A more recent and exciting approach has emerged from the use of endophytic fungi to alter plant responses and adaptation to abiotic stresses.

Scores of studies have examined the role of endophytes in enabling plant adaptation to abiotic stresses. One of the most extensively studied fungi is *Piriformospora*, a root endophyte, isolated from woody shrubs of the Thar Desert, India (Varma et al. 1999). Recently, following a taxonomic revision, the fungus has been renamed *Serendipita indica* (Weiß et al. 2016). The fungus readily colonizes a wide array of plants and imparts tolerance to abiotic stresses such as drought, salinity, osmotic and heavy metals (Hoseini et al. 2017). Aside from conferring adaptation to abiotic stresses, *S. indica* colonization of soybean plants was shown to improve plant growth and also nutrient acquisition (Bajaj et al. 2018). Under salinity stress, maize plants colonised by *S. indica* produced higher biomass and maintained higher shoot potassium ion content compared to un-inoculated plants (Yun et al. 2018). Using rice as a model system, studies have shown that endophytic fungi from salt-adapted plants enhance growth and yield of salt sensitive rice varieties under salinity stress when compared to plants not colonized by such fungi (Redman et al. 2011; Yuan et al. 2016). Sangamesh et al. (2017) demonstrated the ability of endophytes isolated from plants adapted to deserts to not only successfully colonize non-host plants such as rice, but also to impart thermo-tolerance to them under laboratory conditions. In a meta-analysis conducted on 94 strains of endophytes and 42 host plants, Rho et al. (2018) reported that, overall, endophyte colonization led to effective mitigation of drought and salinity stress as well as nitrogen deficiency. The study also showed the ability of endophytes to readily colonize and establish plant-endo-phyte relationships. The existing evidences suggest that endophytes from stress-adapted plants could be transferred across plants of varied phylogenetic affiliations.

The immediate physiological and molecular basis of plant-endophyte interactions, including how endophytes from plants adapted to extreme habitats are able to confer resistance to non-host plants, are only beginning to be addressed. A cyclophilin A-like protein (*PiCypA*) obtained from *Serendipita indica* has been implicated in the ability of the fungus to impart salinity tolerance. Transgenic tobacco plants overexpressing *PiCypA* exhibited higher salt stress tolerance compared to wild type plants (Trivedi et al. 2013). A salt responsive gene, *PiHOG1* from *S. indica*, was shown to impart osmotic and salt stress tolerance to rice plants when compared to mutants in-vitro. Treatment of *S. indica* knock down, *KD-PiHOG1* resulted in decreased colonization and reduced tolerance to salt stress (Jogawat

et al. 2016). *Arabidopsis* plants inoculated with *S. indica* exhibited ion homeostasis under salt stress. These plants had higher transcript levels of high affinity potassium transporters, *HKT1* and inward rectifying K⁺ channels, *KAT1* and *KAT2*, compared to plants without the fungus (Abdelaziz et al. 2017). Under both normal and low phosphorous conditions, *S. indica* activated signaling, transport, metabolic and developmental programs in roots of *Arabidopsis* (Bakshi et al. 2017). The fungus was also shown to cause global reprogramming of host metabolic compounds and metabolic pathways in Chinese cabbage (Hua et al. 2017).

It is evident from existing studies that endophytes offer a promising option to mitigate abiotic stresses in crop plants. The single most important advantage of this approach is that it offers a non-genetic-invasive method to alter plant phenotype, when compared to conventional and molecular breeding approaches (Gopal and Gupta 2016); furthermore, it is rapid and cost-effective. Several initiatives have been launched to harness the power of endophytes, including BioEnsure, a product approved for use by the US Food and Drug Administration, and Rootonic, a mixture of *S. indica* biomass and magnesium sulphate (Jones 2013; Shrivastava and Varma 2014). The product Bioensure was able to stabilize yields of maize under drought, increase seed germination under freezing stress several-fold, and improve the water use efficiency of plants (Jones 2013). Rootonic treatment to seeds provided multifarious benefits to the plants, under both normal and stressful conditions (Shrivastava and Varma 2014). Rapid methods of deployment of select endophytes, either through seed priming or through foliar or floral dip methods, could offer a quick and safe agronomic strategy to mitigate abiotic stress in plants (Fig. 23). This approach also resonates in its application to major crop plants that may have lost many of their native endophytes during the process of domestication. For these plants, the endophyte-enrichment approach could in fact be a process of returning to their roots (Oyserman et al. 2018).

## Food and beverages from fungi

Food and beverages from fungi is a multi-million dollar business, and in particular in Asia, much of the development has concentrated on mushrooms because of their nutritious qualities and medicinal importance. In this section, we discuss the rapid development of the mushroom industry in Asia, outline the mushrooms used in Traditional Chinese Medicine, highlight one of the most revered medicinal mushrooms (*Agaricus subrufescens*), and introduce various food products from fungi.
**Fig. 23** A schematic illustration of the events depicting the use of endophytic fungus in mitigating abiotic (salinity) stress in crop plants. Clockwise: A saline adapted plant, *Prosopis juliflora* (1); isolation of endophytes (2); evaluation of endophytic fungus for salinity tolerance (3); priming seeds of non-host, salt sensitive paddy, with saline tolerant endophyte (4); growth of endophyte-enriched and non-enriched seedlings under saline stress (5); growth of enriched and non-enriched paddy plants under saline stress (6); trans-generational effect of paddy plants under saline stress (7). The model could be followed for other abiotic stresses as well (all photographs: School of Ecology and Conservation, University of Agricultural Sciences, GKVK, Bangalore)

<figure><img src="image_34.png" /></figure>

## 23. Growing mushrooms in composts

Some mushrooms require a composting process for industrial production, as in *Agaricus* species. The methods for growing *Agaricus* species differ from those used in growing many other mushrooms, which are grown in sawdust mixtures in bags. *Agaricus* is a secondary decomposer. The preparation of the substrate for growing *Agaricus* species is a process known as composting. Within this process, bacteria and other fungi break down the raw materials in the compost mixture, which allows *Agaricus* species to grow on the fermented substrate (Llarena-

Hernández et al. 2011). The first formal mention of mushroom cultivation based on the composting method was in 1650 in Paris. However, cultivated mushrooms were reported to be different in appearance to those harvested from the field and not as good to eat. In 1707, a French botanist reported that mushrooms were produced from horse manure covered by soil. The first record of commercial cultivation was in 1780 by a French gardener, while mushroom growing was introduced to North America after the Civil War (Beyer 2003). In 1800, the first cultivation of *Agaricus* in caves was achieved in Paris.
In Europe and Brazil, the basic raw material used in composting is wheat straw. hay, corncobs, oat, barley, sugarcane bagasse, rye grass, rice straw, several other grasses or spent mushroom waste can act as alternative substrates (Royse and Chalupa 2009; Stamets and Chilton 1983; Mendonça et al. 2005; Llarena-Hernández et al. 2011, 2014; Grimm and Wösten 2018). Supplements such as soybean meal and cereal bran mixed with minerals or vitamins are added for cultivation of *Agaricus* and *Pleurotus* mushrooms (Carrasco et al. 2018). There are normally two phases in composting. Phase I is called composting, which involves mixing the piled-up ingredients. Supplements are added to the watered stack and this is incubated for 3–15 days. The temperature inside the compost stack can reach 70–80 °C, and thermophilic microbes in the compost utilize carbohydrates and free ammonia. After fermentation, the compost is pasteurized at 60 °C. Phase II is called conditioning, at which point the temperature is maintained at 45–60 °C for the culturing of good microbes, and to decrease the ammonium level of the compost (Mendonça et al. 2005; Stamets and Chilton 1983; Grimm and Wösten 2018).

In Asia composting has been modified as wheat straw and horse manure are not readily available. Chopped rice straw is used as the main substrate. The rice straw is supplemented with rice bran, urea, ammonium phosphate, calcium sulfate to increase nutrients. Gypsum and calcium carbonate are added for buffering the pH. The process occurs outdoors. For pasteurization, the compost is heated to 55–60 °C for 3–6 h with steam (Royal project's method, unpublished data). When the compost is generally used and inoculated with spawn. After the mycelium runs through the compost the casing is covered to stimulate fruit-body production (Llarena-Hernández et al. 2013). Different mixtures are used for casing including soil: wood, charcoal: calcitic lime (Zied et al. 2010), soil: sand mixed with peat (Mendonça et al. 2005) limestone: peat: thin sand (Llarena-Hernández et al. 2014) after casing. Then, the mycelium grows within the casing layer in about 15–20 days depending on environmental factors, *Agaricus* requires around 20–30 days (Mendonça et al. 2005).

Methods of cultivation have been developed that use local agricultural waste as the main substrate, instead of wheat or rice straw. Specific media need to be established for the production of fruit-bodies depending on the species of mushroom.

## Mushrooms grown in compost

### *Agaricus bisporus* (Button mushroom)

The first white cultivar of *Agaricus bisporus* was found in 1926 by Lewis Downing, an American farmer, who

passed it on to a spawn company. Four year later, the spawn had been spread worldwide. This cultivar named “smooth white” is one of parents of the hybrid strain most cultivated in the world. The basic cultivation method for *A. bisporus* was established and developed by Sinden and Hauser (1950, 1953). *Agaricus bisporus* can be grown on standard compost based on wheat straw and horse manure, as well as other agricultural waste (Colak 2004; Carrasco et al. 2018). Peat, sand, peat and lime (1:1:1); farm yard manure and soil (1:1); tea production waste mixed with peat; and protein-rich supplements (soybean, black beans and cowpeas) can be used as a casing layer (Gülser and Pekşen 2003; Coello-Castillo et al. 2009; Gupta et al. 2018) *Agaricus bisporus* can also be cultivated using autoclaved non-composted substrates (Till 1962; Colmenares-Cruz et al. 2017).

### Other *Agaricus* species

*Agaricus flocculosipes* was first reported by Zhao et al. (2012) and is currently only known to be found in Thailand, Mayotte Island and China. Thongklang et al. (2014a) reported the first successful cultivation of *A. flocculosipes* in Thailand on wheat straw and horse manure compost. The primordia of Thai *A. flocculosipes* occurred at day 32 after casing, and the strain produced a few fruiting bodies (Fig. 24). *Agaricus subtilipes* (Fig. 24) was described in Thailand by Thongklang et al. (2016). Cultivation of *A. subtilipes* was successful on wheat compost with fruiting bodies produced 45 days after casing. Although *A. flocculosipes* and *A. subtilipes* produced fruiting bodies, the yields were low and the fruiting period was shorter when compared to Thai strain of *A. subrufescens* (Fig. 24). These Thai *Agaricus* species are candidates for domestication and cultivation and have potential medicinal use. But first the optimal conditions for growing these mushrooms must be established, and any bioactive compounds investigated. In addition, the optimal conditions for fruiting must be established for increasing yield production. Hyde et al. (2018b) reported that 93% of *Agaricus* species found in northern Thailand were new to science and, as many are edible, they have high potential for domestication.

### *Pleurotus ostreatus* (oyster mushroom)

This mushroom is cultivated worldwide, grows easily in various substrates, and has important nutritional and medicinal properties. The oyster mushroom is a primary decomposer and can degrade many components of plant material, including hemicellulose, lignin and more (Grimm and Wösten 2018). The mushroom is produced on rubber sawdust, rubber sawdust mixed with different supplements of rice straw, rice husks or corncobs in Thailand via bag cultivation (Thongklang and Luangharn 2016). Vieira and de Andrade (2016) reported that compost was used to cultivate *P. ostreatus* in Brazil. Different substrates,