Journal of global issues & solutions (May-June 2015)

http://www.bwwsociety.org/journal/current/applied_biology.htm

Directors letter for the journal of global issues & solutions (May-June 2015 issue):

From the sphere of Innovation: Applied Biology, Professor Dinh Xuan Ba, Director of the SECOIN
Applied Biology Center in Hanoi, Vietnam presents his paper entitled Vietnamese Farmer Uses Ants to
Create Rare Aloeswood. I visited Professor Ba at his company headquarters in Hanoi in 1996 and
again in 2009; during my second visit I was stuck at the tremendous growth his company had experienced
during that thirteen year period. It was at that time that Professor Ba explained to me his passion for
applied biology and the important research work he conducts in the area of Sapa, in the very northern
region of Vietnam. Modern Vietnam is an exciting country experiencing rapid growth, with more than a
decade of consecutive year-over-year increases in its economy. My passion for Vietnam began with my
first visit to this Southeast Asian nation, during which I witnessed the countrys invigorating enthusiasm
and an impressively innovative and creative populace. In terms of opportunity, Vietnam is where the
United States was 100 years ago: young, vital, strong and expanding, and with an incredible growth of a
new middle-class that was practically non-existent 20 years ago. One of the phenomena of this rapid
growth is that of numerous farmers who were operating at bare subsistence level in 1990s who are (US
dollar) multi-millionaires today simply because what had been farmland has become prime residential
and commercial real estate. However, the Vietnamese farmer who is the focus of Professor Bas paper
has become a success not through real estate, but through to his persistence in searching for a method to
create the very rare substance, aloeswood. http://www.bwwsociety.org/journal/current/director0515.htm)

Innovation: Applied Biology:

Vietnamese Farmer Uses Ants to Create Rare Aloeswood

by Prof. Dinh Xuan Ba
Director, SECOIN Applied Biology Center
Hanoi, Vietnam

Aloeswood (Agarwood), or tram huong as called by the Vietnamese, is a species of plant favored
by Vietnamese and Asian countries because of its special fragrance. Tram huong, or agarwood,
is a resinous heartwood, used for perfume, aromatherapy, alternative medicine and incense.

Due to the depletion of natural raw materials, first-grade agarwood is one of the most expensive
natural raw materials in the world. Since 1995 some Aquilaria species have been listed as
potentially threatened species by the Convention on International Trade in Endangered Species
of Wild Fauna and Flora. The current global market for agarwood is estimated to be in the range
of US$6-8 billion and is growing rapidly.
Mr. Tran Duy Linh, a noted Vietnamese herbalist, has been quoted as saying that tram huong can
be used to cure various diseases, particularly digestive problems, and help improve virility. It is
very expensive, with one kilogram priced at tens of thousands of US dollars.

With resources in short supply and prices putting the product out of range for a substantial
number of consumers, a strong market for cultivated agarwood has evolved over the last 20
years. Challenges have been encountered in finding an effective method by which to produce
cultivated agarwood.

Then, in 2010, a Vietnamese farmer, Mr. Truong Thanh Khoan, developed a method of
cultivating a very high quality agarwood from Aquilaria trees. Mr. Khoans techniques are
sufficiently unique that he was granted a patent on the method he has developed.

Mr. Khoan said that he had spent 17 years looking for tram huong in the wild, but had failed
because the plants had become more scarce due to unfavorable habitat conditions. This being the
case, he decided to attempt to create tram huong himself.

At first, Mr. Khoan planted young Do Bau (Aquilaria crassna) in his garden. When the trees
grew to a diameter of 10-15 cm, he injected chemicals into the trunks, hoping to create artificial
aloeswood. However, the method was not successful. He then decided to stimulate the fragrance-
creation process by injecting a mixture of molasses, plant nutrient media and yeast extracts.
Although the biological product was safe and helpful to the development of tram huong, it still
was unsatisfactory.

Then, by chance, Mr. Khoan came upon a wounded tree trunk with ants nests inside the wounds.
It was a surprise to him that the veins around the ants nests looked shiny-black, similar to the
color of tram huong in the wild. Seeing this, Mr. Khoan decided to breed ants to create tram
huong.

Ants produce a substance which helps the process of forming tram huong on Aquilaria
Agallocha (Do Bau) more quickly than any artificial method, Mr. Khoan said about the role of
the ants. Ants eat plants and produce a liquid which has a color similar to that of molasses. The
liquid, if mixed with molasses, bee honey, maize bran and coconut oil extract, will help speed up
the fermentation process which allows it to create biological products.

When the biological product is injected into wounds on Do Bau plants, the plants produce a
kind of antibiotic to cover the wounds and to fight bacteria, yeasts and fungi. The creation of
this antibiotic is a crucial condition to the successful cultivation of tram huong.
Mr. Khoan has utilized his practical discovery on a production area of five hectares with over
3,000 Do Bau plants under cultivation. Beyond the technical value of Mr. Khoans innovation,
there also lies a heart-warming human interest story, wherein this simple farmer is en route to
becoming a multi-millionaire through his creativity, perspicacity and persistence.

A detailed scientific explanation of Mr. Khoans work is provided:

The Innovation of Cultivating Agarwood Using Ant-Fungus Mutualism

Preface

On April 20, 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 June 9,
2014, the National Office of Intellectual Property of Vietnam (under the Ministry of Science and
Technology) granted Mr. Khoan Patent Number 12835 for his Inducement Method on Aquilaria
trees for creating cultivated Agarwood (fig.3). Unlike all inducers previously existing in Vietnam
and around the world, Mr. Khoans inducer contains quintessential nectar produced by ants,
hereinafter referred to as ant nectar. Mr. Khoan spent 20 years in the forest, not only looking for
natural Agarwood, but also to painstakingly search for inducers; his success was limited. Only
upon his discovery of a species of ant (nesting in Aquilaria tree) that has the ability of biological
stimulation for Agarwood formation, was he set upon a successful path. He began to study the
ants living habits in order to domesticate them, and went on to create appropriate conditions for
them, so that the ants can produce the ant nectar that is the key component of Mr. Khoans
special inducer (referred to as Ant-Processed Inducer, or ApI). Based on this ApI, Mr. Khoan
has succeed in creating Agarwood in thousands of Aquilaria trees (fig.4, fig.5, fig.6) and he now
has become a wealthy peasant intellectual. On September 12, 2011, he applied to the Ministry of
Science and Technology for an exclusive patent protection of his invention. Mr. Khoans ApI
inducer has been successfully utilized in numerous locations throughout Vietnam and has been
promoting in the international marketplace.

The purpose of this article is to explain the scientific basis of this invention. If we can point out
scientific foundation of this discovery, we then can alter this unique creative achievement of
practical 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 review some basic knowledge as follows:

What is cultivated agarwood ?

Association of ant-fungus mutualism
What is Cultivated Agarwood?
Agarwood is comprised of diseased tissues located in the heartwood of several varieties of some
genera of the family Thymelaeaceae. The main source of Agarwood is the Aquilaria tree, of
which 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 (for example, one
kilogram of natural agarwood can be worth tens, and even 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 and pathological infection created by artificial inducement
- The floristic composition of vegetation 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. The artificial inducement technique (or Agarwood inducement) consists of two
actions: to wound the tree and then to pour a stimulative agent into this injury; this stimulative
agent is referred to as inducer. The quantity and quality of cultivated agarwood surrounding the
injury depends upon the composition of the inducer and the method of transfusing the inducer
into the tree.

Currently, there are dozens of inducers, the composition of which is kept secret. Generally
speaking, the main components of inducer are:
- Some fungi and microfungi, for example: Fusarium
oxysporum Schlecht., Cladosporium spp, Cercosporella spp,Aspergillus Phoenicis (Cord
a) Thom & Currie, Cytosphaera Manganiferae Died, Melanotus flavolivens (Berk. &
M.A. Curtis) Singer, Penicillium Citrinum Thom, Chaetonium
globosum Kunze, Phaeoacremonium parasitica and others. Of these, Fusarium,
Cladosporium and Cercosporella are three of 17 fungi that were found in nine natural
agarwood samples collected from forests in Thailand (3), while Fusarium and Chaetonium
are two fungi found in natural agarwood pieces collected in India (4).
- Some phytochemicals, namely: formic acid, methyl jasmonate, chitosan, sodium
bisulfite, and others.
- Some bacteria, yeast extracts, phytohormones, plant nutrient media and plant regulators

There are two primary methods of transfusing inducer into the Aquilaria tree:
 - Dispersed wounding by mechanical injury: to drill several dozen (20-100) holes into the
tree, 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 a transfusion set; by this method only two to four holes are needed (fig.9). If the
internal wounding technique to be applied, then induced agarwood is formed in the entire
tree along the xylem (fig.10).

Association of Ant-Fungus Mutualism

Ants live everywhere on Earth, but most live in tropical regions; there are more than 13,000
species of ants with about 10,000,000 billion individual ants. Ants vary in color; most are red or
black, but a few species are green (or green moss color) and some tropical species have a
metallic luster. Ants live in colonies and a perfect division of labor exists amongst them. Ants
have a fascinating social structure, in that they are eusocial insects. Within an ant colony there
are the 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 about ants and the scientific
study of ants (myrmecology) came into the world.

In this presentation we will focus attention on the following ant communities that are living in
the tropical and subtropical rainforests: Fungus-growing ants (fig.11), Carpenter ants (wood
ants) (fig.12) and Weaver ants (green ants) (fig.13).

Ants created farming (or the practice of agriculture) about 50 million years ago (5):

- Ants can practice animal husbandry, specifically, ants can breed aphids. The aphid is
a myrmecophile (the term myrmecophile is used mainly for animals and insects that live
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 provides an abundant food source for ants (aphids of 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 comprised of various sugars, while the remaining
matter includes vitamins, minerals and amino acids. Ants live in association with other
honeydew-producing insects such as Coccidae, Pseudococcidae, Membracidae and
others. There are also many other myrmecophiles and organisms that coexist with ants in
their colonies, namely: fungi and yeasts, microfungus Escovopsis, bacteria, and so on.(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 could not survive without the fungus. This is the mutualism
between ant-cultivated fungi and fungus-growing ants.
- Worker ants, which are all female, are divided into two types, media and minima, both
of which have large 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, the slightly smaller minima workers cut up the
leaves into small pieces and then feed it to a fungus they cultivate. The minima workers
also act as guards and attack small parasitic flies; the minima workers follow the media
workers or hitch a ride on the leaf theyre carrying to the source of the leaves. The ants
use an antibiotic-producing actinomycete bacteria, that grows on the ants, as an antibiotic
against any invasive molds; this is how they keep their nests 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. The average diameter of
gongylidium is about 40 microns. Gongylodia occur in clusters in the 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, we need to consider the two following questions: 1.
How many species of fungus-growing ants exist and to what genera do they belong? And 2.
What are the names and types of ant-cultivated fungi?

In 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 the 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),
and Trachymyrmex Forel (48 species). The genera Atta Fabricius and Acromyrmex Mayr have
the common name Leaf-cutting Ant (fig.15). We are willing to share basic biological data of
above mentioned 237 ant species.

In 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 the Leucoagaricus genus, Leucocoprinus genus and Leucoagaricus gongylophorus
species are frequently seen (7).
Nevertheless, in regard to the topic of this presentation, we will pay attention to the following
scientific findings:

In 2008, Rodrigues A and colleagues (9) revealed a total of 85 microfungal strains, of

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
genusCladosporium 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. It
can also be found in Agarwood inoculant created by the AIPA (Agarwood Inoculant Producer
Association) of Indonesia (fig.17).

About Ant-processed Inducer (ApI)

In 2010 Mr. Truong Thanh Khoan started to domesticate a species of ant that nests in Aquilaria
tree. He made about 20 wooden cages, with one ant colony per each cage. He grows vegetable
for ant forage and provides the ants with coconut milk.

Is it true that Khoans ant variety is one of 49 above-mentioned species of leaf-cutting ants?

These ant cages were designed to easily gather the gongylidia which is squeezed and filtered to
obtain a thick solution referred to as ant nectar. Ant nectar is a key component of Ant-processed
Inducer (ApI), and each liter of ApI contains 10ml of ant nectar. The invention of ApI (fig.18)
was made public on the internet, via http://agarwood.ning.com(11) , and in print publication via
Trang Trai Viet No. 41 November 2014 (pp28-31)(12). An ApI Research & Development team
was recently formed in Vietnam (fig.19), the primary missions of which 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 of domesticated ants, including
the Ophiocordyceps fungus that creates zombie-ants(13). (fig.20)
4. Biochemical analysis of gongylidia and any nectar 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 nectar from which to find an
appropriate ant tendance for increasing gongylidia biomass.
 6. Improvement of the 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 compounds 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 a marketing network for ApI and cultivated agarwood.

Important note: We, ApI R&D team, hereby call for help and cooperation from myrmecologist,
chemist and biologist for consolidating scientific ground of this innovation.

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, Maurcio
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.0103800
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.http://agarwood.ning.com/profiles/blogs/an-invention-of-agarwood-inducement-created-
by-a-vietnamese?xg_source=activity
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-
amazon and http://ento.psu.edu/directory/dph14
14. Forest pathology- http://en.wikipedia.org/wiki/Forest_pathology
Further reading: http://youtube/qHSIYmsPM28

Contact details:

Mr. Truong Thanh Khoan; Phone: 01234699679. E-mail: dan.tr88@gmail.com.

Prof. Dinh xuan Ba; Phone: 0913207676. E-mail: dxb@secoin.vn.
Mr. Nguyen Binh Quang Chinh (English name: Peter); Phone: 093 748 1989. E-
mail: sales@secoin.vn.
Ms. inh Mai Ly (English name: Miley); Phone: 093 564 4458. E-mail: miley@secoin.vn

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