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What is Agarwood inducement (continued)

Internal wounding - alteration


What is Agarwood inducement (continued)

If the

internal

wounding

technique

to be

applied

then

induced

agarwood

is formed

in whole

tree along

the xylem.


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).


Association of ant-fungus mutualism (continued)

Winged alates mate

Workers



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

play attention to the following ant communities that are living in the

tropical and subtropical rainforests: Fungus-growing ants, Carpenter

ants (or wood ants) and Weaver ants (or green ants).

Fungus-growing ant

Weaver ant (green ant) Carpenter ant (wood ant)



Other pictures of fungus-

growing ants, carpenter ants

and weaver ants.

Fungus-growing ant

Carpenter ant (wood ant)

Weaver ant (Green ant)


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. 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 created farming from about 50 million years ago (5), namely:



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. 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 abour 40

microns. Gongylodia occur in clusters in

fungus garden where ant queen stays.

Mueller UG and colleagues (5) isolated

553 fungus cultivars from gardens of

fungus-growing ants.



Fungus garden



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. We are willing to

share basic biological data of above mentioned 237 ant species.

Acromyrmex striatus Acromyrmex echinatior Apterostigma auriculatum



Atta laevigata Cyphomyrmex rimosus

Mycocepurus curvispinosus Myrmicocrypta worker

Sericomyrmex amabilis Trachymyrmex septentrionalis



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).

The white

material in the

picture

probably is the

fungus

Leucocoprinus

gongylophorus

(http://www.mar

ietta.edu/~biol/

costa_rica/ani

mals/leafcutter

_ants.htm)



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

These two findings are specially significant because Fusarium oxysporum

and Cladosporium spp are two fungi found in natural agarwood pieces

collected from Thailand and India. Also can find it in Agarwood inoculant

made by AIPA (Agarwood Inoculant Producer Association) of Indonesia.


About Ant-processed Inducer

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 260 above-mentioned species of fungus-growing ants ?


About Ant-processed Inducer (continued)

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 was made public in

http://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.

http://agarwood.ning.com/









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). Zombie-ant Ophiocordyceps



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 (TLC: Thin Layer

Chromatography).



8. Evaluation of volatile aromatic compound

in cultivated agarwood: Using GCMS

chromatograms of cultivated essential oils to

identify and estimate the quantity of typical

sesquiterpenes and derivatives (GCMS: Gas

Chromatograph Mass Spectrometer).

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.

















References (continued)

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.010

3800

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.


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

http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103800





















http://agarwood.ning.com/profiles/blogs/an-invention-of-agarwood-inducement-created-by-a-vietnamese?xg_source=activity

























References (continued)

12. Dinh xuan Ba –Thuoc cay tao Tram tu “dich kien”. Trang trai Viet No. 41

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

Referential addresses: Mr. Truong thanh Khoan; No. 3478, Phu Lam 3 hamlet, Phu Son commune, Tan Phu

district, Dong Nai province. Tel: 01234699679. E-mail: [email protected].

Mr. Truong Tuan Vu; No. 3478, Phu Lam 3 hamlet, Phu Son commune, Tan Phu

district, Dong Nai province. Tel: 0965565639. E-mail: [email protected].

Prof. Dinh xuan Ba; Villa KL41, Kim Long villa complex, Nha Be district, HoChiMinh

city. Tel: 0913207676. E-mail: [email protected]

Mr. Tran duc Thanh; No.54/4, Ngoc Lam 3 hamlet, Phu Thanh commune, Tan Phu

district, Dong Nai province. Tel: 0985561610. E-mail:

[email protected].

Ms. Nguyen thi Huyen Tran; No.54/4, Ngoc Lam 3 hamlet, Phu Thanh commune, Tan

Phu district, Dong Nai province. Tel: 0978653359. E-mail:

[email protected]

THANK YOU

http://www.theguardian.com/science/2011/mar/02/fungi-zombie-ants-amazon








http://ento.psu.edu/directory/dph14

http://en.wikipedia.org/wiki/Forest_pathology

mailto:[email protected]























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