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Isolation and identification of Endophytic Fungus Fusarium sp from Agarwood

Isolation and identification of Endophytic Fungus Fusarium sp from Agarwood ( Aquilaria sp ) population originated from the forest of Aceh Tamiang district, Indonesia

Aquilaria sp is one of higher plant types that can produce agarwood. Agarwood is commonly stimulated by the biological response of endophytic fungal infection of Fusarium sp . This research aims to isolate and identify endophytic fungus Fusarium sp from Aquilaria sp populations originated from the forest of Aceh Tamiang District. The pure isolates of Fusarium sp derived from Aquilaria sp that reinfected on Aquilaria sp populations to stimulate agarwood formation. The endophytic fungal isolates of Fusarium sp were growth on a selective media of Sabouraud Dextrose Agar (SDA) containing antibiotics Streptomycin 10 µg/dl and Chloramphenicol 25µg/dl. Ten percent of infected Aquilaria sp from the population was taken aseptically. Isolation and identification of endophytic fungus Fusarium sp were conducted from June to August 2016. The results showed that within 10 infected Aquilaria sp , several microbe species were found such as three Fusarium sp , six Aspergillus sp , two Rhizopus sp , two Penicillium sp , one Bacillus sp , one Actinomyces sp and one Streptomyces sp species. The result of identification towards three species of endophytic fungus Fusarium sp that has been purified were srongly suspected to be Fusarium solani, Fusarium oxysporum , and Fusarium monyliformae.
The wood fibre containing agarwood from sampling techniques, a; sample of wood fibre containing agarwood in sterile adhesive plastic, b; endophytic fungus from xylem, c; endophytic fungus from wood fibre of Aquilaria sp.

The wood fibre containing agarwood from sampling techniques, a; sample of wood fibre containing agarwood in sterile adhesive plastic, b; endophytic fungus from xylem, c; endophytic fungus from wood fibre of Aquilaria sp.

The endopyhtic fungal isolates of Fusarium solani, Fusarium oxyforum and Fusarium monyliformae grown on SDA media, A; Fusarium oxyforum grown on SDA media, incubation at room temperature, day 5 th incubation, B; Fusarium oxyforum grown on SDA media, incubation at room temperature, on day 7 th of incubation.

The endopyhtic fungal isolates of Fusarium solani, Fusarium oxyforum and Fusarium monyliformae grown on SDA media, A; Fusarium oxyforum grown on SDA media, incubation at room temperature, day 5 th incubation, B; Fusarium oxyforum grown on SDA media, incubation at room temperature, on day 7 th of incubation.


The endophyte fungal sampling location on some Aquilaria sp populations on four different locations in Aceh Tamiang Regency.

The endophyte fungal sampling location on some Aquilaria sp populations on four different locations in Aceh Tamiang Regency.


Endophytic fungal and bacteria isolates isolated from ten stems of Aquilaria sp from four different forest in AcehTamiang.

Endophytic fungal and bacteria isolates isolated from ten stems of Aquilaria sp from four different forest in AcehTamiang.


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Agarwood induced


Agarwood is broadly used in incense and medicine. Traditionally, agarwood formation is induced by wounding the trunks and branches of some species of Aquilaria spp., including A. sinensis. As recently evidenced, some fungi or their fermentation liquid may have the potential of inducing agarwood formation. The present study aimed to analyze the fungi isolated from an agarwood-producing A. sinensis tree and subsequently identify the fungi capable of promoting agarwood formation. We identified a total of 110 fungi isolates based on their morphological characteristics and rDNA ITS sequences. These isolates came from four different layers (namely the decomposing layer, agarwood layer, transition layer, and normal layer) near the agarwood formation site of the trunk. According to the experimental results, most of them belonged to Dothideomycetes (81.82%), while the others to Sordariomycetes (13.64%) or Eurotiomycetes (4.55%). Of note, 88 isolates were shown belonging to the species of Lasiodiplodia theobromae that are most frequently isolated from different layers. In addition, when the fermentation liquid of two isolates of L. theobromae (AF4 and AF12) and one isolate of Fusarium solani (AF21) was inoculated into the A. sinensis wood using the Agar-Wit technique, promoted agarwood formation was observed; however, the effect of AF21 did not keep stable in the later test, while AF4 and AF12 still functioned 1 year later. This study may lay a foundation for exploring the underlying mechanism of agarwood formation as well as fungi application in agarwood production.

Trầm hương được sử dụng rộng rãi trong hương liệu và y học. Theo truyền thống, sự hình thành trầm hương được tạo ra bằng cách làm tổn thương thân và cành của một số loài Aquilaria spp., bao gồm cả A. sinensis. Như đã được chứng minh gần đây, một số loại nấm hoặc chất lỏng lên men của chúng có thể có khả năng tạo ra trầm hương. Nghiên cứu hiện tại nhằm mục đích phân tích các loại nấm phân lập từ cây A. sinensis sản xuất trầm hương và sau đó xác định loại nấm có khả năng thúc đẩy sự hình thành trầm hương. Chúng tôi đã xác định được tổng cộng 110 chủng nấm phân lập dựa trên đặc điểm hình thái và trình tự rDNA ITS của chúng. Các chủng phân lập này đến từ bốn lớp khác nhau (cụ thể là lớp phân hủy, lớp trầm hương, lớp chuyển tiếp và lớp bình thường) gần vị trí hình thành trầm hương của thân cây. Theo kết quả thực nghiệm, hầu hết chúng thuộc về Dothideomycetes (81,82%), còn lại thuộc Sordariomycetes (13,64%) hoặc Eurotiomycetes (4,55%). Đáng chú ý, 88 chủng phân lập được cho thấy thuộc loài Lasiodiplodia theobromae thường được phân lập từ các lớp khác nhau. Ngoài ra, khi chất lỏng lên men của hai chủng L. theobromae (AF4 và AF12) và một chủng Fusarium solani (AF21) được cấy vào gỗ A. sinensis bằng kỹ thuật Agar-Wit, người ta đã quan sát thấy sự hình thành trầm hương được thúc đẩy; tuy nhiên, hiệu quả của AF21 không giữ được ổn định trong lần thử nghiệm sau đó, trong khi AF4 và AF12 vẫn hoạt động được 1 năm sau đó. Nghiên cứu này có thể đặt nền tảng cho việc khám phá cơ chế hình thành trầm hương cũng như ứng dụng nấm trong sản xuất trầm hương.

Agarwood induced by the branch-cutting method was used for fungi isolation in the present study. Four layers were separately collected. DL decomposing layer, AL agarwood layer, TL transition layer, NL normal layer

Agarwood induced by the branch-cutting method was used for fungi isolation in the present study. Four layers were separately collected. DL decomposing layer, AL agarwood layer, TL transition layer, NL normal layer.

Agarwood is broadly used in incense and medicine. Traditionally, agarwood formation is induced by wounding the trunks and branches of some species of Aquilaria spp., including A. sinensis. As recently evidenced, some fungi or their fermentation liquid may have the potential of inducing agarwood formation. The present study aimed to analyze the fung...
The numbers of isolated strains from different layers. The species of Lasiodiplodia theobromae was dominantly spreading in each layer

The numbers of isolated strains from different layers. The species of Lasiodiplodia theobromae was dominantly spreading in each layer.

Agarwood induced by the fermentation liquid of isolated fungi. a Wood inoculated with: 1 the fermentation liquid of NAF6; 2 culture media; 3 the fermentation liquid of AF12; 4 the fermentation liquid of AF4; 5 the patented agarwood inducer; 6 the fermentation liquid of AF21. b Plants that were inoculated with the fermentation liquid of fungi showed yellow leaves; c colony of AF4; d colony of AF12; e colony of NAF6; f colony of AF21; g thin-layer chromatogram of extracts from the wood inoculated with: 1 the fermentation liquid of AF21; 2 the patented agarwood inducer; 3 the fermentation liquid of AF4; 4 the fermentation liquid of AF12; 5 ST; 6 7-dimethoxy-2-(2-phenylethyl) chromone, and six culture media. (Color figure online)

Agarwood induced by the fermentation liquid of isolated fungi. a Wood inoculated with: 1 the fermentation liquid of NAF6; 2 culture media; 3 the fermentation liquid of AF12; 4 the fermentation liquid of AF4; 5 the patented agarwood inducer; 6 the fermentation liquid of AF21. b Plants that were inoculated with the fermentation liquid of fungi showed yellow leaves; c colony of AF4; d colony of AF12; e colony of NAF6; f colony of AF21; g thin-layer chromatogram of extracts from the wood inoculated with: 1 the fermentation liquid of AF21; 2 the patented agarwood inducer; 3 the fermentation liquid of AF4; 4 the fermentation liquid of AF12; 5 ST; 6 7-dimethoxy-2-(2-phenylethyl) chromone, and six culture media. (Color figure online).


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Artificial inoculation



人工處理植入催化劑

Artificial inoculation technique already been developed and is found to be most effective and reliable method for enhancement of agar production. Works on commercialization are in progress and expected to make available for general use in the field.

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 inoculant. Quantity and quality of cultivated agarwood surrounding the injury depends on the composition of inoculant and the method of transfusing inoculant into the tree. 

Nowadays, there are about several tens of inoculants whose composition is kept secret. But in general the main components of inoculant are:

  • Some fungi and microfungi, for example: Fusarium oxysporum Schlecht., Cladosporium spp, Cercosporella spp, Aspergillus Phoenicis (Corda) Thom & Currie, Cytosphaera Manganiferae Died, Melanotus flavolivens (BerPlant hormonesk. & 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, Fusarium and Chaetonium are two fungi found in natural agarwood pieces collected from India. 
  • Some phytochemicals, phytohormones namely: salicylic acid, ethephon, methyl jasmonate, chitosan, sodium bisulfite,..
  • Some bacteria, yeast extracts, plant nutrient media and plant regulators 

Two main methods Agarwoodof transfusing inoculant into Aquilaria tree: 

Dispersing wounding by mechanical injury: to drill several tens (20-100) of hole into which the inoculant is poured. In this case induced agarwood is formed around these spot injury sites. The resulting agarwood from the localized formation is usually in the form of thicker wood chips.

Internal wounding: to transfuse inoculant directly into tAgarwoodhe xylem vessels of tree by using transfusion set, in this case only few holes are needed. If the internal wounding technique to be applied then induced agarwood is formed in whole tree along the xylem, from root/base up to the top of the trunk in a systemic manner, but relatively thinner formation of resinous wood. 

Lately, inoculant operators have adopted to Dispersing wounding, leaving the Internal wounding for few reasons after gaining numerous field experiences … wood rotting is the main drawback.

gaharu

wood rotting by internal wounding

While internal wounding is preferred by certain people, it can be further improved with “Pressurized method”. The air pressure can be portably pumped range 0.5 ~ 1.5 bar, hence minimize local concentration which resulting in tissue rotten. For cost effective, unit can be connected to multiple points, and reusable in next application. This invention is simple, inoculation process completed at faster time, no ingress of rain water even the injection holes are not sealed. Click “Mini pump“.

Pressurized method

Dispersing wounding, mechanically wounded with nails, traditional way in Bangladesh

Dispersing wounding, tree drilling and chemical injection.

DSC_4217-Edits800

This effort involves drilling of trees and keeping the wound open by placing a small piece of plastic pipe in those holes followed by a chemical injection to stimulate tree defense mechanism that produces resin. The first project of this effort was initiated in Vietnam under a supervision from Prof. Robert Blanchette, a wood pathologist from the University of Minnesota who together with local farmers and Buddhist monks. They developed experimental plots to stimulate the production of agarwood and after years of trials, this treatment yielded agarwood. Dr. Blanchette stated that this artificial induction could yield agarwood ten times faster than natural formation (WWW page: http://forestpathology.coafes.umn.edu). This finding has gained appreciation and has been considered as one of the most successful finding (Persoon, 2007). This treatment causes tree to respond in two defense mechanisms, physical and chemical. The first is phloem cells form callus and the second, should callus formation is prevented, the tree produces resin.

Dispersing wounding with 10~20ml liquid inoculant, some inoculant service providers utilize upto 100ml per treated hole.

Dispersing wounding 1Dispersing wounding 2Dispersing wounding 3Dispersing wounding 4

Dispersing wounding, inoculant-soaked bamboo stick, Thailand
CIMG0467

Dispersing wounding, inoculation of fungal inoculum6158739645_8725dbb2b6_b

The formation of agarwood is a result of plant defense mechanism towards fungal attacks by producing resinous compounds as secondary metabolite. In their natural habitat, the process of resin accumulation as the result of tree-pathogen battle may take many years and the longer the process takes place the more expensive and highly valuable the resin is. Many scientists have been passionately trying to understand the cascade process of this tree-fungi interaction in producing gaharu. Isolation of various fungi from infected trees have been widely reported.

Surface Inducement Technique ( Agar-SIT )… inoculant is designed to brush on the tree trunk. This technique allows easy agarwood collection, a considerably high yield and good resinous of agarwood could be harvested, along with the sustainable agarwood formation from the same tree. Specifically, this technique is aimed for oil production. Click “Agar-SIT-105” for detail …

trunk surface inoculantTrunk stree

Dispersing wounding, non-destrustive harvest, by Pheeraphan Thailand

Thai dispersing 1

gaharu

agarwoodagarwood

Thai dispersing 2Thai dispersing 3

Thai dispersing 4

The infected tissue is stripped by chisel

Internal wounding, Single unit, 300ml inoculant per tree,  by PENGHARUM.

Internal wounding, Multiple units, by Gaharu Biotech,Pahang.

Picture2

Gravitically flow.

wounding

Application of Chemjet, pressure exerted 0.75bar. Test plot to verify strength of inoculant

Inoculant on Sinensis Click Here

Artificial fire method, China人工火烧法It is to burn the live agarwood tree with a large fire, forcing the agarwood tree to be injured. It looks very cruel, but it does not let the agarwood tree die, so the agarwood tree will mobilize the resin secreted by the immune system to heal the fire, and eventually form agarwood. Select the older tree, peel off the bark from the tree, leave the top leaves, and then use  gas torch to burn the whole tree and burn it black. This method is derived from the natural fire or the process of lightning strikes. The agarwood produced in this way is mostly thin, it is used a lot to produce incense.

inoculationIn China, newer species of Kynam has been successfully grafted, this species requires mechanical stress without any injection of liquid inoculant to produce resinous agarwood. Application of soldering device is sufficient for the job. However, the seedlings demand extremely premium price for the moment, click “Kynam

kynam23.jpgkynam24

 

 

 

 

 

 

 

Planting money (Agarwood) trees doesn’t come without its challenges. One of these challenges is the inoculation. The Aquilaria tree has to be induced with a infection to induce production of agarwood (the resin), otherwise the tree would be quite worthless. Not all Aquilaria trees produce agarwood, they have to be infected by a certain kind of stress before they start “fighting back” with resin production to counter the infection. And even so, there is no set volume in the resin production (meaning each tree will produce its own amount).




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