An invention of forming cultivated agarwood using ant-fungus mutualism

[Dinh xuan Ba]

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An invention of forming cultivated agarwood using ant-fungus mutualism

Prof. Dinh xuan Ba
Director of SECOIN Applied Biology Center

Preface
What is cultivated agarwood ?
Association of ant-fungus mutualism
About Ant-processed Inducer

Freface

On 20th April 2014, The Vietnam Intellectual Property Association granted to Mr. Truong thanh Khoan ( fig.1), a Vietnamese farmer, a “trustworthy product” certificate on his inducer applied to Aquilaria trees and on cultivated Agarwood formed by using this inducer (fig.2). On 9th June 2014, The National Office of Intellectual Property of Vietnam (under The Ministry of Science and Technology) gave Mr. Truong Thanh Khoan a patent number 12835 about Inducement method on Aquilaria trees for creating cultivated Agarwood (fig.3). Unlike all inducers presently existing in Vietnam and around the world, Mr. Khoan’s inducer contains quintessential juice produced by ants, hereinafter referred to as “ant juice”. Mr. Khoan formerly spent 20 years in the forest, not only to seek for natural Agarwood, but also to gropingly look for inducers but his result was limited. Only after the day where he discovered a species of ant (nesting in Aquilaria tree) that has the ability of biological stimulation for Agarwood formation, he started to study their living habits in order to domesticate them and to create approriate conditions for them, so that they can produce ant juice that is a key component of a special inducer herinafter to be called as Ant-processed inducer (ApI). Based on this ApI, Mr. Khoan has succeed in creating Agarwood in many thousands of Aquilaria trees (fig.4, fig.5, fig.6) and now he has became a rich peasant intellectual, a Vietnamese billionaire. Right from September 12th 2011, he applied to The Ministry of Science and Technology for soliciting an exclusive protection of his invention. His ApI inducer has been successfully applied to numerous locations in Vietnam and has been being promoting to foreign countries. The purpose of this article is to try explaining scientific ground of this invention. If we can point out scientific foundation of this discover, we then can alter this unique creative achievement of pratical into an intellectual fortune, we also can assign to this experiential finding a professional language, therefore we can develop this product inside and outside of Vietnam. To do that, we need to know some basic knowledges as follows:
• What is cultivated agarwood ?
• Association of ant-fungus mutualism

What is cultivated agarwood ?

Agarwood is diseased tissues located in the heartwood of several trees of some genera of the family Thymelaeaceae, the main source of Agarwood is the Aquilaria trees (there are 26 species of the genus Aquilaria that can produce Agarwood). Natural Agarwood is an aromatic resinous wood that is extremely rare and expensive (one kg of natural agarwood worth several hundreds of thousands of US dollars). According to Robert A. Blanchette (1) and Pheeraphan Wijitphan (2) (fig.7), Agarwood is a pathological product of plant defense (or immune response) against outside attacks and impacts, namely:
- Naturally pathological infection caused by fungi and other herbivores (insects, bacteria,..)
- Mechanical injury impacted by human being or outer factors (war, storm, ..)
- Wounding & pathological infection created by artificial inducement
- The floristic composition of vegetational cover (companion plants, insects,..) may possibly play a certain role in Agarwood formation.

The Agarwood created by artificial inducement is called Cultivated Agarwood or Induced Agarwood. Artificial inducement technique (or Agarwood inducement) consists of two actions: to wound the tree and to pour stimulative agent into this injury, this stimulative agent hereinafter referred to as inducer. Quantity and quality of cultivated agarwood surrounding the injury depends on the composition of inducer and the method of transfusing inducer into the tree.

Nowadays, there are about several tens of inducers whose composition is kept secret. But in general the main components of inducer are:
- Some fungi and microfungi, for example: Fusarium oxysporum Schlecht., Cladosporium spp, Cercosporella spp, Aspergillus Phoenicis (Corda) Thom & Currie, Cytosphaera Manganiferae Died, Melanotus flavolivens (Berk. & M.A. Curtis) Singer, Penicillium Citrinum Thom, Chaetonium globosum Kunze, Phaeoacremonium parasitica,…in which Fusarium, Cladosporium and Cercosporella are three of 17 fungi that were found in 9 natural agarwood samples collected from the forest in Thailand (3), Fusarium and Chaetonium are two fungi found in natural agarwood pieces collected from India (4).
- Some phytochemicals, namely: formic acid, methyl jasmonate, chitosan, sodium bisulfite,..
- Some bacteria, yeast extracts, phytohormones, plant nutrient media and plant regulators

Two main methods of transfusing inducer into Aquilaria tree:
- Dispersing wounding by mechanical injury: to drill several tens (20-100) of hole into which the inducer is poured (fig.8). In this case induced agarwood is formed around these injury sites (fig.5, fig.6).
- Internal wounding: to transfuse inducer slowly and directly to the xylem vessels of tree by using transfusion set, in this case only two or four holes are needed (fig.9). If the internal wounding technique to be applied then induced agarwood is formed in whole tree along the xylem (fig.10).

Association of ant-fungus mutualism

Ants live in everywhere on the Earth, but most live in tropical regions and there are more than 13,000 species of ants with about 10,000,000 billion individual ants. Ants vary in colour; most ants are red or black, but a few species are green (or green moss color) and some tropical species have a metallic lustre. Ants live in colonies and a perfect division of labor exists amongst them, they have a pretty interesting social structure, in other words ants are eusocial insects. In the ant colony there are queen, princess, workers and soldiers. An ant colony can be established under a single queen (monogyny) or under multiple queens (polygyny). Ants are a very important part of ecosystems and in the study of biodiversity. In 1874 the first book on the ants and the scientific study of ants (myrmecology) came into the world. In this presentation we pay attention to the following ant communities that are living in the tropical and subtropical rainforests: Fungus-growing ants (fig.11), Carpenter ants (or wood ants) (fig.12) and Weaver ants (or green ants) (fig.13).

Ants created farming (or practice agriculture) from about 50 million years ago (5), namely:
- Ants can practice “animal husbandry”, for ínstance ants can breed aphids, in other words aphid is a myrmecophile (the term myrmecophile is used mainly for animal that lives in mutualistic association with ants). Aphids feed on the phloem sap of plants and excrete honeydew droplets. The tending ants ingest these honeydew droplets (fig.14). Aphid honeydew can provide an abundant food source for ants (aphids in the genus Tuberolachnus can secrete more honeydew droplets per hour than their body weight). Between 90-95% of the dry weight of aphid honeydew is various sugars, while the remaining matters includes vitamins, minerals and amino acids. Ants are in associations with other honeydew-producing insects such as Coccidae, Pseudococcidae, Membracidae…There are also a lot of other myrmecophiles and organisms that coexist with ants in their colonies, namely: fungi and yeats, microfungus Escovopsis, bacteria,…(6)
- Ants can practice “agriculture” for example ants can cultivate fungi. Ants cut and process fresh vegetation (leaves, flowers, and grasses) to serve as the nutritional substrate for their fungal cultivars (fig.15). Fungus-growing ants cultivate fungi as their most important food source and in turn the fungus is nourished, protected against harmful microorganisms, and dispersed by the fungus-growing ants. The fungus could not survive without the ants, and the ants cannot survive without the fungus. This is a mutualism between ant-cultivated fungi and fungus-growing ants. For details: Ant workers, who are all female, are divided into two types, media and minima, that both have big jaws and sharp teeth. Media workers are the larger of the two, and they cut the leaves and bring them back to the nest. Once the media workers have deposited the material into the nest, slightly smaller workers called ‘minima workers’ cut up the leaves into small pieces and then feed it to a fungus they cultivate. These minima workers also act as guards and follow the media workers or hitch a ride on the leaf they’re carrying to the source of the leaves and attack small parasitic flies. But the ants use the antibiotic producing actinomycete bacteria, that grows on the ants, as an antibiotic against any invasive molds. This is how they keep their nest so clean and disease free. The plant material is broken down through enzymes that break down the proteins and starches that are nutrients for the ants, which may accumulate in specialized hyphal-tips known as gongylidia. According to a latest research result of Virginia E. Masiulionis and colleagues(7) : Gongylidia usually consists of glucose, glycogen, glycan, mannitol, trehalose, lipid, ergosterol, enzymes and free amino acids. Average diameter of gongylidium is about 40 microns. Gongylodia occur in clusters in fungus garden where ant queen stays (fig.16). Mueller UG and colleagues (5) isolated 553 fungus cultivars from gardens of fungus-growing ants.

Concerning the subject of this presentation, two following questions are needed to make clear: Firstly, how many species of fungus-growing ants and to what genera they belong ? Secondly, names and types of ant-cultivated fungi ?

The answer to the first question: There are 260 species of fungus-growing ants that belong to 18 genera of the tribe Attini. It is recommended to pay attention to 8 main genera with 237 species as follows: Acromyrmex Mayr (32 species), Apterostigma Mayr (47 species), Atta Fabricius (17 species), Cyphomyrmex Mayr (41 species), Mycocepurus Forel (6 species), Myrmicocrypta Smith (27 species), Sericomyrmex Mayr (19 species), Trachymyrmex Forel (48 species). The genus Atta Fabricius and Acromyrmex Mayr has the common name Leaf-cutting Ant (fig.15). We are willing to share basic biological data of above mentioned 237 ant species.

The answer to the second question: According to Augustin JO and colleagues (8): Ant-cultivated fungi usually belong to the families Agaricaceae, Lepiotaceae and Pterulaceae, in which the fungi of Leucoagaricus genus, Leucocoprinus genus and Leucoagaricus gongylophorus species are frequently seeing (7).

Nevertheless, in regard to the topic of this presentation, we are paying attention to the following scientific findings:

In 2008, Rodrigues A and colleagues (9) revealed a total of 85 microfungal strains, in which Fusarium oxysporum was the predominant species in the surveyed fungus gardens, infecting 40.5% of 37 surveyed nests. Also in this year, Pagnocca FC and colleagues (10) isolated from the body parts of leaf-cutting ants 142 filamentous fungi and 19 yeasts, in which the genus Cladosporium prevailed 78% among filamentous fungi.

These two findings are specially significant because Fusarium oxysporum and Cladosporium spp are two fungi found in natural agarwood pieces collected from Vietnam, Thailand and India. Also can find it in Agarwood inoculant created by AIPA (Agarwood Inoculant Producer Association) of Indonesia (fig.17).

About Ant-processed Inducer (ApI)

From 2010 a Vietnamese farmer Truong Thanh Khoan started to domesticate a species of ant that nests in Aquilaria tree. He made about 20 wooden cages, one ant colony per each cage. He grows vegetable for ant forage, gives ants coconut milk as drink,….Is it true that Khoan’s ant is one of 49 above-mentioned species of leaf-cutting ants ?
These ant cages were designed for easy gathering gongylidia which is squeezed and filtered to obtain a thick solution named “ant juice”. Ant juice is a key component of Ant-processed Inducer (ApI), each liter of ApI contains 10ml of ant juice. The invention of ApI (fig.18) was made public in https://agarwood.ning.com/(11) and in Trang Trai Viet No. 41 November 2014 (pp28-31)(12). One R&D team of ApI is recently formed in Vietnam (fig.19). Primary missions of this R&D team are:
1. Identification of the ant species to be domesticated by Mr. Khoan (scientific name, genus, species, phylogenetic classification,…).
2. Minute investigation of association ant-fungus mutualism, including microorganisms living symbiotically, commensally and parasitically in ant colonies.
3. Identification and prevention of diseases and enemies for domesticated ants, including Ophiocordyceps fungus that creates zombie-ants(13). (fig.20)
4. Biochemical analysis of gongylidia and “ant juice” from which to study the negative relations of domesticated ant colonies with forest pathogens(14).
5. Quantitative study of the yield of gongylidia and “ant juice” from which to find an appropriate ant tendance for increasing gongylidia biomass.
6. Improvement on appropriate constituents of ApI and in methods of transfusing ApI into Aquilaria trees.
7. Evaluation of fragrant oleoresin in cultivated agarwood: Using TLC chromatograms of cultivated agarwood to identify typical chromones derivatives of natural agarwood
8. Evaluation of volatile aromatic compound in cultivated agarwood: Using GC/MS chromatograms of cultivated essential oils to identify and estimate the quantity of typical sesquiterpenes and derivatives.
9. Diversification of cultivated agarwood products obtained by using ApI including agarwood pieces, decorative arts, essential oils, incenses, aromatic oleoresins,…
10. Promotion of marketing network for ApI and cultivated agarwood.

References

1. United States Patent No. US 6,848,211 B2 – Feb. 1, 2005
2. United States Patent No. US 7,485,309 B1 – Feb. 3, 2009
3. Subansenee W., Naiyana Tongjiem, Viboon Sakekul (1985) "Fungi on agarwood [Aquilaria spp.]." Report on minor forest products research, Royal Forestry Dept., Bangkok (Thailand). Forest Product Research Div.- Bangkok (Thailand), 1985. p. 8-15.
4. Tamuli P, Boruah P, Nath SC, & Samanta R (2000) “Fungi from diseased agarwood tree (Aquilaria agallocha Roxb.): two new records”, Advances in Forestry Research 2000, XXII ed. Ram Parkash p182-189.
5. Mueller UG, Rehner SA, Schultz TR. The evolution of agriculture in ants. Science. 1998 Sep 25; 281(5385):2034-8. http://www.ncbi.nlm.nih.gov/pubmed/9748164/
6. Carreiro SC, Pagnocca FC, Bueno OC, Bacci M Junior, Hebling MJ, da Silva OA. Yeasts associated with nests of the leaf-cutting ant Atta sexdens rubropilosa Forel. Antonie Van Leeuwenhoek. 1997 Mar; 71(3):243-8. http://www.ncbi.nlm.nih.gov/pubmed/9111918/
7. Virginia E. Masiulionis, Christian Rabeling, Henrik H. De Fine Licht, Ted Schultz, Maurício Bacci Jr, Cintia M. Santos. Bezerra, Fernando C. Pagnocca. A Brazilian Population of the Asexual Fungus Growing Ant Mycocepurus smithii (Formicidae, Myrmicinae, Attini) Cultivates Fungal Symbionts with Gongylidia-Like Structures. Published: August 07, 2014. http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone....
8. Augustin JO, Groenewald JZ, Nascimento RJ, Mizubuti ES,…Yet more "weeds" in the garden: fungal novelties from nests of leaf-cutting ants. PLoS One. 2013 Dec 20; 8(12):e82265. http://www.ncbi.nlm.nih.gov/pubmed/24376525
9. Rodrigues A, Bacci M Jr, Mueller UG, Ortiz A, Pagnocca. FC. Microfungal "weeds" in the leafcutter ant symbiosis. Microb Ecol. 2008 Nov; 56(4):604-14. http://www.ncbi.nlm.nih.gov/pubmed/18369523
10. Pagnocca FC, Rodrigues A, Nagamoto NS, Bacci M Jr. Yeasts and filamentous fungi carried by the gynes of leaf-cutting ants. Antonie Van Leeuwenhoek. 2008 Nov; 94(4): 517-26. http://www.ncbi.nlm.nih.gov/pubmed/18665453
11. Dinh xuan Ba. An invention of agarwood inducement created by a Vietnamese farmer. https://agarwood.ning.com/profiles/blogs/an-invention-of-agarwood-i...
12. Dinh xuan Ba –Thuoc cay tao Tram tu “dich kien”. Trang trai Viet No. 41- trang 28-31
13. David P. Hughes – Ian Sample. Fungi that create 'zombie ants' discovered in Brazilian jungle. http://www.theguardian.com/science/2011/mar/02/fungi-zombie-ants-am... and http://ento.psu.edu/directory/dph14
14. Forest pathology- http://en.wikipedia.org/wiki/Forest_pathology
Further reading: http://youtu.be/qHSIYmsPM28

Referential addresses:

Mr. Truong thanh Khoan; No. 3478, Phu Lam 3 hamlet, Phu Son commune, Tan Phu district, Dong Nai province. Tel: [phone]. E-mail: [e-mail].  
Mr. Truong Tuan Vu; No. 3478, Phu Lam 3 hamlet, Phu Son commune, Tan Phu district, Dong Nai province. Tel: [phone]. E-mail: [e-mail].  
Prof. Dinh xuan Ba; Villa KL41, Kim Long villa complex, Nha Be district, HoChiMinh city. Tel: [phone]. E-mail: [e-mail].
Mr. Tran duc Thanh; No.54/4, Ngoc Lam 3 hamlet, Phu Thanh commune, Tan Phu district, Dong Nai province. Tel: [phone]. E-mail: [e-mail].
Ms. Nguyen thi Huyen Tran; No.54/4, Ngoc Lam 3 hamlet, Phu Thanh commune, Tan Phu district, Dong Nai province. Tel: [phone]. E-mail: [e-mail].

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