Taxonomy and biology of calonectria in forest soil of vietnam

Basing on data of 72 soil samples which were collected from some forests of Vietnam such as Hoang Lien National Park, Tam Dao National Park, and Binh Phuoc province, the study clearly de

ACKNOWLEDGEMENTS

In order to complete this study course, with the consent of the Department of Forest

Resources and Environment Management (FREM), I conduct to do a research: “Taxonomy

and biology of Calonectria in forest soil of Vietnam”

In the implementing research process, I have received the helps of Forest Protection Research Centre (FPRC) - Vietnamese Academy of Forest Sciences (VAFS), everyone who work in this centre, and especially the enthusiastic guidance of Associated Professor Ph.D Phung Van Khoa - Vietnam National University of Forestry and Associate Professor Ph.D Pham Quang Thu - Forest Protection Research Centre

Therefore, I would like to express deep gratitude to Associated Professor Ph.D Phung Van Khoa, Associated Professor Ph.D Pham Quang Thu who guide for me Thanks to Forest Protection Research Centre, and all members in here who have created favorable conditions for

me to complete this study I also have a good eye for helping of my family, and my friends

In the process of learning and doing research, I tried my best but time, knowledge, and budget are limited Therefore, this study could not avoid some faults I am looking forward to hearing comments from you to more complete the study

ABSTRACT

The research presents taxonomy and biology of Calonectria in forest soil of Vietnam

Basing on data of 72 soil samples which were collected from some forests of Vietnam such as Hoang Lien National Park, Tam Dao National Park, and Binh Phuoc province, the study

clearly described biology characteristics and pathogenicity of Calonectria Furthermore,

taxonomy also is an important part of this research Using method of sample collection in collecting soil samples; method of isolation, culturing the fungus in finding fungi phylogeny; method of pathogenicity in defining pathogenic ability; method of DNA technique in fungi identification; method of using microscope in describing morphological characteristics of fungi; and methods of culturing the fungus in different environment, temperature, moisture to determine the biological characteristics of fungi Data was collected in 2016 from 72 soil samples in Hoang Lien National Park, Tam Dao National Park, and Binh Phuoc province The results of the study showed morphological characteristics and biological characteristics

of 41 isolates- not only this but also they were classified Generally, this pathogenic fungi need to be paid more attention in order to protect forest trees

TABLE OF CONTENTS

ACKNOWLEDGEMENTS

ABSTRACT

TABLE OF CONTENTS

LIST OF FIGURES

LIST OF TABLES

1 INTRODUCTION 1

1.1 Imperativeness 1

1.2 Study site 1

1.3 Object of research 5

1.4 Literature reviews 5

2 GOALS AND (SPECIFIC) OBJECTIVES 8

2.1 Goals 8

2.2 Specific objectives 8

3 METHODS 9

3.1 Sampling methods: 9

3.2 Isolation methods: 10

3.3 Methods of pathogenicity testing: 11

3.4 DNA technique: PCR amplification and sequencing: 13

3.5 Method of describing the morphological characteristics of fungi: 14

3.6 Method of describing the biological characteristics of fungi: 15

3.6.1 Growth of the mycelium on different nutrient media: 15

3.6.2 Growth of the mycelium on PDA nutrient medium at different temperature scales: 16

3.6.3 Growth of the mycelium on PDA nutrient medium at different moisture scales: 17

4 RESULTS 18

4.1 Result of sample collection 18

4.2 Result of isolation, and culturing the fungus 19

4.3 Pathogenicity testing 22

4.4 Taxonomy by DNA technique: PCR amplification and sequencing 24

4.5 Morphological characteristics of fungi 28

4.5.1 Morphological characteristics of Ca follicola (DMC TN2.1) 28

4.5.2 Morphological characteristics of Ca pentaseptata (RTN BPP3.3) 28

4.5.3 Morphological characteristics of Ca illicicola (LT DN 2.1 & LT DN2.2) 29

4.6 Biological characteristics of fungi 29

4.6.1 Growth of the mycelium on different nutrient media 29

4.6.2 Growth of the mycelium on PDA nutrient medium at different temperature scales: 32

4.6.3 Growth of the mycelium on PDA nutrient medium at different moisture scales 33

5 DISCUSSION 37

6 CONCLUSION 39 REFERENCES

LIST OF FIGURES

Figure 1.1 Study site 2

Figure 3.1 Soil samples 9

Figure 3.2 Process of isolation and culturing the fungus 11

Figure 3.3 Confront sample and pathogenicity sample 12

Figure 3.4 Microscope and camera use to describe morphological characteristics 15

Figure 3.5 Three types of nutrient media 16

Figure 3.6 Fungus culturing 16

Figure 3.7 Moisture experiment 17

Figure 4.1 Some strains under stereoscope 19

Figure 4.2 Spore of Calonectria 20

Figure 4.3 Four groups of isolates 22

Figure 4.4 Pathogenic ability of fungal strains 23

Figure 4.5 The process of pathogenicity of Calonectria on Eucalyptus leaf 23

Figure 4.6 Classification tree of DMC TN2.1 25

Figure 4.7 Classification tree of RTN BP3.3 26

Figure 4.8 Classification trees of LT DN2.1, and LT DN2.2 27

Figure 4.9 Vesicle and spore of Ca follicola 28

Figure 4.10 Vesicle and spore of Ca pentaseptata 29

Figure 4.11 Vesicle and spore of Ca illicicola 29

Figure 4.12 Growth of the mycelium on different nutrient media 30

Figure 4.13 Difference growth of mycelium in OSA, PDA, and MEA nutrient medium 31

Figure 4.14 Growth of some mycelium in different temperatures 32

Figure 4.15 Growth of mycelium at different temperature scales after 3 days 33

Figure 4.16 Growth of some mycelium in different moisture after 3 days 34

Figure 4.17 Growth of some mycelium in different moisture after 5 days 35

LIST OF TABLES

Table 1.1 Calonectria species reported in Vietnam 7

Table 3.1 Levels of pathogenic ability 13

Table 3.2 Formula to create moisture environment 17

Table 4.1 List of soil samples is collected 18

Table 4.2 List of fungi strains were found 19

Table 4.3 Some characteristics of 41 fungi strains 20

Table 4.4 Pathogenic strains and pathogenic levels of them 24

1 INTRODUCTION 1.1 Imperativeness

In Vietnam, Cylindrocladium quinqueseptatum, anamoph of Calonectria species firstly was found from Hue to the South as fungal pathogens to Eucalyptus spp Calonectria

pentaseptata was announced in 2012 by Pham Quang Thu and coworkers (L.Lombard,

M.J.Wings, & Crous, sp nov) And now, this fungus was found in most area of Vietnam Therefore, I conducted this research to find out the diversity and characteristics of

Calonectria Hope that this study will be useful with disease management in the future in

order to protect flora

1.2 Study site

As we know, Vietnam is located in the Indochina peninsula, belongs to South-East Asia region The Northern part of Vietnam is bordered by China; Cambodia and Laos in the West; the Southwest is bordered by the Gulf of Thailand; East and South, Vietnam is bordered by the East Sea Vietnam has an area of 331 698 km², of which three quarters are hilly In Vietnam, about 327 480 km² is terrestrial, and 4,500 km² is an inland sea Because Vietnam is located along the coast, the climate is regulated in part by sea currents, so it brings more marine climate factors Average relative humidity is 84% throughout the year Every year, precipitation is from 1,200 to 3,000 mm, the number’s hours of sunshine is about 1,500 to 3,000 hours / year, and the temperature is between 5°C to 37°C

With conditions such as terrain and climate were presented, Vietnam is a good place for the development of diverse animals and plants, especially the forest fauna, and forest flora As is already known, mainly Vietnam area is hilly Thus, we can see the forest has a very important role in Vietnam However, with hot, humid climate conditions and heavy rainfall in some areas, these are favorable conditions for pests and harmful fungi grow In this

study, 3 sites with topographic features, the climate in Vietnam was chosen to study are

Hoang Lien National Park, Tam Dao National Park, and Binh Phuoc province

Figure 1.1 Study site

First location is Hoang Lien National Park This is a national park within Hoang Lien Son Range It has total area 28.509 ha, located in 4 communes of Sapa district (Lao Chai, Ta Van, San Sa Ho, Ban Ho) in Lao Cai province and 2 communes (Phuc Khoa and Trung Dong) of Tan Uyen district, Lai Chau province This National Park has many differences compared to other national parks in the special use forest (SUF) system of Vietnam where the exchange of the two subtropical climates and alpine temperature happens Terrain, this is where is rugged terrain, high slopes, fragmented, peaks above 1,000 meters, including Fansipan with 3.143m high For specific characteristics about climate, weather, topographic, Hoang Lien has incredibly rich of fauna – flora According to the scientists, Hoang Lien National Park is one of the most diverse biodiversity centers Vietnam, especially flora There exist here over 2,000 species of plants typically as Bo tree, azaleas, plum…, of which about

66 species are recorded in Vietnam’s Red Book, 32 rare species, and hundreds of precious herbs Besides, several ancient mushroom species were also detected here

Second location is Tam Dao National Park which is a protected area zone in North Vietnam The park is about 85km northwest of Hanoi It runs 80 km from North- West

to South East, and has more than 20 peaks with altitudes of over 1000m The highest summit

is Tam Dao North with an altitude of 1592m Three other peaks with beautiful scenery are Thien Thi at 1375m, Thach Ban at 1388m, and Phu Nghia at 1300m Sharp peaks with sloping sides and numerous, deep partitions are characteristic of the topology Due to the tall mountainous range that splits the area into two parts, the national park's climatic condition is divided into two areas with different rainfalls According to research, Tam Dao park has eight kinds of forest types distributed in different topographic and climatic areas : the tropical moist evergreen forest, the subtropical moist evergreen low mountain forest, the high mountain short forest, the bamboo forest, the restored forest, the plantation forest, the bush appears, the grass land Out of over 2000 plant species, 904 species are considered useful to

humans There are 42 species endemic to Tam Dao National Park and also 64 other species considered rare The national park has diverse animal species, with 840 species in total Eleven of these species are endemic to Tam Dao National Park including the snake species, the amphibian and eight species of insects; 22 species are endemic to North Vietnam including nine bird species, four reptiles, three amphibians and six species of insect; six are

endemic to Vietnam (five bird species and one species of amphibian)

Last location is Binh Phuoc province whichis located in the Southeast region of the country, to the north of Ho Chi Minh City It shares a border with Cambodia Binh Phuoc Province is relatively flat with elevations of between 50m and 200m throughout most of the provinces Elevations are gradually higher towards the east of the province and reach around 500m near parts of the border to Dak Nong Province of the Central Highlands The highest elevation is Ba Ra Mountain (736m) in the centre of the province There are several hills around the province with heights of up to around 200m in the west and 300m in the southeast Forestry land takes up 337,000 ha or 49 per cent of the province's total area Forests are located mostly in the northeast and southeast of the province as well as along the northern border to Cambodia and the western border of Tay Ninh Province Much of the rest of the area is used to grow perennial cash crops The total agricultural area is 293,700 ha There is 21,900 ha of specially used land and 5700 ha of residential land Average annual rainfall in the province is 2280 mm / year, varying from 1900 to 2731 mm / year, is unevenly distributed In general, rainfall increases from the Southeast to the Northwest, in which the area has average rainfall less than 2000 mm is 115,093.7 hectares (16.83%), an area with average rainfall of 2,000 mm to 2500 mm is 301,964.3 hectares (44.2%), an area with average rainfall of 2500 mm is greater than 266,666.3 ha (39%) The average annual temperature is 26°C, ranges from 24.3°C to 28°C December has lowest average monthly

temperature (24.3°C) April has maximum average monthly temperature (28°C), unevenly distributed, tend to decrease from Southwest to Northeast

1.3 Object of research

As we know, flora is the most important part in forest ecosystems It not only reflects the richness of the genetic biodiversity of forest resources, but also determines the diversity of forests, scientific value, the value of landscape and environmental, economic benefit All above locations have suitable conditions for growth of flora However, high temperature, heavy rainfall was also favorable conditions for pests and diseases development, including a

pathogenic fungus –Calonectriawhich is object of this study This is one kind of serious

pathogenic fungus in plant which we need to care if we want to protect the growth of tree in there

Most of Calonectria species are readily recovered from soil (Crous, 2002) Therefore, this

study also conducts isolate them from soil samples

1.4 Literature reviews

The genus Calonectria (Ca.) was erected in 1867 by De Notaris, based on Ca

daldiniana collected on leaves of Magnolia grandiflora that belong to Magnoliaceae, in Italy

(Rossman 1979a) Then Rossman (1979a) reduced Ca daldiniana to synonymy under Ca

pyrochroa This nectrioid fungus was defined as having an ascocarp wall structure that is

brightly coloured, changing to blood-red in 3% KOH solution, warty to scaly and with a

Cylindrocladium (Cy.) anamorph (Rossman 1993, Rossman et al 1999) However, due to the

restricted morphological characteristics of the teleomorph (Rossman 1979b, 1983), specimens can in many cases only be identified to species level if the anamorph is present

(Schoch et al 2000b, Crous 2002) (L Lombard et al, 2010)

The anamorph genus Cylindrocladium, which is based on Cy scoparium, was first

described by Morgan (1892) in the U.S.A., where it was found growing as saprobe on a pod

of Gleditsia triacanthos Although Morgan (1892) failed to mention the stipe extension terminating in a vesicle of characteristic shape, he defined the genus as having branched conidiophores producing cylindrical conidia This fungus has a wide distribution in sub-tropical and tropical regions of the world, and species are pathogenic to numerous plants

(Crous 2002) (L Lombard et al, 2010)

Lombard also showed that Calonectria belongs to the Nectriaceae, one of three

families in Hypocreales, an order that has been reviewed extensively The Nectriaceae is circumscribed as having uniloculate ascomata that are orange to purple and not immersed in well-developed stromata The family includes approximately 20 genera of socio-economic

importance and Calonectria is most clearly distinguished from the others by its

Cylindrocladium anamorphs and relevance as plant pathogens

The genus Calonectria was initially regarded as a saprobe as no disease symptoms

could be induced by inoculating a suspected host The first proof of pathogenicity of these fungi was provided by Massey in 1917, and subsequently by Anderson in 1919, who proved

pathogenicity of Ca morganii (as Cy scoparium) Then, Calonectria species have been

associated with a wide range of disease symptoms on a large number of hosts worldwide In

the past, several authors have indicated that Calonectria species cause disease on plants

residing in approximately 30 plant families (Booth & Gibson 1973, French & Menge

1978, Peerally 1991a, Wiapara et al 1996, Schoch et al 1999) Upon closer inspection, the

number of plant families is actually closer to 100 and approximately 335 plant host species The plant hosts include important forestry, agricultural and horticultural crops and the impact

of these plant pathogens has likely been underestimated (L Lombard et al, 2010)

The majority of disease reports associated with Calonectria species in forestry include hosts in 5 plant families, of which the most important are associated with Fabaceae (Acacia spp.), Myrtaceae (Eucalyptus spp.) and Pinaceae (Pinus spp.) The symptoms may include

leaf spot, stem lesion, stem or crown canker, root rot, crown rot and stem rot In Vietnam,

Ca.insularis and Ca pauciramosa were also recorded for the first time, where they are

associated with leaf spot symptoms on Eucalyptus spp (Crous P.W et al, 2002)

Table 1.1 Calonectria species reported in Vietnam

FUNGAL

SPECIES

DISEASE

Ca insularis Leaf spot Eucalyptus sp Crouset al 2002

Ca pauciramosa Leaf spot Eucalyptus sp Crouset al 2002

Ca reteaudii Leaf blight Eucalyptus spp Booth et al 2000

Old et al 2003

Ca pentaseptata Leaf blight Eucalyptus hybrid Crouset al 2012

Stemming from this situation, I conducted a study "Research on Taxonomy and

Biology of Calonectria in forest soil of Vietnam" The aim of this research is to isolates and identification of fungal species in the genus Calonectria through DNA sequence analysis and

morphological characteristic, and identifying, researching biological characteristics, pathogenicity of isolation Hopefully, this study is useful in order to manage plant diseases

and the conservation and development of flora biodiversity in Vietnam

2 GOALS AND (SPECIFIC) OBJECTIVES 2.1 Goals

This study has two main points: Taxonomy and Biology We also have 2 main goals:

- In Taxonomy: to identify the fungal species in the genus Calonectria through DNA

sequence analysis and morphological characteristic

- In Biology: to identify and study biological characteristics, and pathogenicity ability

3 METHODS 3.1 Sampling methods:

The disease is often compromised in the root; stem and fungal sources usually exist in

the soil And most of Calonectria species are readily recovered from soil (Crous, 2002)

Extensive surveys will be taken in study sites As was mentioned above, in Vietnam,

Ca.insularis and Ca pauciramosa were also recorded with leaf spot symptoms on Eucalyptus

spp (Crous P.W et al, 2002) Therefore, we conduct to collect soil sample from Eucalyptus

plantations, and many difference places Trying to find the place where the soil was not dry

for the Calonectria spp likes the moist environment

The soils samples will be collected from positions having symptomatic plants, natural forest soil will also conclude Soil samples will be kept in plastic bags, marked and well-informed about the time and place of sampling, bring samples back to laboratory for assessment

Figure 3.1 Soil samples

 Spray bottle of distilled water

 PDA nutrient medium (formula for 1 liter): 200g potatoes minced put in 1L water Boiling it in 30 minutes, filter the water Then add enough water to 1 liter of water Thereafter, add 20g glucose and 18g agars, shake well to dissolve, steamed for 20 minutes at 121°C Lastly, waiting to temperature of solution is about 50°C pouring them into petri dishes

With antibiotic PDA nutrient medium, we add 0.2g streptomycin into 100ml distilled water, shake well to dissolve Put them into solution of potatoes, agar, and glucose when temperature of solution is about 50°C Then, shake again and pouring them into petri dishes

Conducting:

 Put soil sample into the cup (about 10mm)

 Spray moderate sterilized water into the cup; make sure the surface of the soil sample is moist (moisture is enough)

 Sow the sterilized alfalfa seeds evenly on the soil sample; make sure the seeds

is not crowded, finally cover the lid, put the cup into a sealed chamber

Figure 3.2 Process of isolation and culturing the fungus

After 2 to 4 days, the seeds will germinate, you can check it with a stereoscopic,

microscope If there has Calonectria in the soil, you may find Calonectria infects the alfalfa seeds or immature stems Then you can isolate the Calonectria Using the sharp needle to

pick up marcoconidiophore Then transfer them into antibiotic PDA nutrient medium Subculture them into the PDA nutrient mediumto have pure fungal samples

3.3 Methods of pathogenicity testing:

Pathogenicity test was conducted in leaves of Eucalyptus

Preparing:

 Fungi samples were pure in the petri dish;

 Nest box lined with moist paper;

 Leaves of Eucalyptus tree have similar size in one tree

 Instruments to implant fungus: knives, alcohol lamps, alcoholic, roll bandage, the grid scale

Conducting:

 Putting leaves facing down into nest box lined with moist paper

 Disinfecting knife by alcohol lamps, alcoholic

 Cutting fungi in nutrient medium in similar size plugs, put them on the back of the leaves; using roll bandage to sealed dish

 Tracking and measuring results after 4 days: using the grid to measure diseased leaf area of the total leaf area

Pathogenic ability = The percentage of diseased leaf area =

x100 (%) Note: For control, doing similar but use nutrient medium plug without fungi

Figure 3.3 Confront sample and pathogenicity sample

According to Pham Quang Thu about Eucalyptus disease and disease management (2005) - having self adjustment to suitable, pathogenic ability is divided in 4 levels:

Table3.1 Levels of pathogenic ability Level Pathogen ability Disease leaf area

3.4 DNA technique: PCR amplification and sequencing:

According toL Hywel-Jones (2004), the isolation protocol of Lee & Taylor (1990) was used to isolate genomic DNA from fungal mycelia We also use it in this research

Template DNA (approximately 20ng) was amplified in a 25 (.il PCR reaction mixture consisting of 1 x PCR reaction buffer (10 mM Tris-HCl, 1.5 mM MgCl2, 50 mM KC1, pH 8.3) and 200 uM each of dATP, dCTP, dGTP and dTTP, with primer pairs EF1-728F and EF2were used to amplified fragment of translation elongation factor gene region, and 1.25 units Taq DNA polymerase enzyme (Roche Diagnostics GmbH, Mannheim, Germany) The reaction was set up as follows: initial denaturation at 96°C for 5 min, followed by 30 cycles

of denaturation at 94°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 90 s, and final extension at 72°C for 7 min in a GeneAmp PCR System 2700 (PerkinElmer, Norwalk, Connecticut)

A negative control, in which water was used instead of template DNA, was set up for each experiment PCR products were separated by electrophoresis at 75 V for 1 h in a 0.8% (w/v) agarose gel in 0.5 xTAE buffer (0.4 M Tris, 0.05 M NaAc, 24©Verlag Ferdinand Berger & Söhne Ges.m.b.H., Horn, Austria, download under www.biologiezentrum.at and 0.01 M EDTA, pH 7.85) and visualised under UV light using a GeneGenius Gel

Documentation and Analysis System (Syngene, Cambridge, UK) following ethidium bromide staining PCR products were purified by using a NucleoSpin Extract 2 in 1 Purification Kit (Macherey-Nagel GmbH, Germany) The cycle sequencing reaction with 20 to 40 ng of purified PCR products and 10 pmol primers in a total volume of 10 |.il was carried out with

an ABI PRISM BigDye Terminator v3.0 Cycle Sequencing Ready Reaction Kit (PE Biosystems, Foster City, CA, USA) containing AmpliTaq DNA Polymerase The reaction was set up as denaturing at 94°C for 5 min., followed by 25 cycles of 96°C for 10 s, 55°C for

10 s, and 60°C for 4 min., with a final incubation of 30 s at 60°C

The resulting fragments were analyzed on an ABI Prism 3100 DNA Sequencer (Perkin-Elmer, Norwalk, Connecticut) Then identification taxonomy of the isolates by the DNA sequence analysis

3.5 Method of describing the morphological characteristics of fungi:

Morphological or phenotypic characters have played a major role in the description of fungal species (Brasier 1997, Taylor et al 2000) and form the basis of new fungal descriptions as required by the ICBN (McNeill et al 2005) In recent years, the use of morphological characters alone to delimit new species has been set aside to a large extent, with more focus being placed on biological and phylogenetic characters (Rossman 1996,

Brasier 1997, Taylor et al 2000) This trend is also evident in recent studies on Calonectria

species (Crous et al 2004b, 2006a) Observe the morphological characteristics of the fungus under the microscope Note the characteristics were seen to describe shape, bulkhead, and the size Then, using camera to measure the length of the spores and get pictures

Characteristics of the anamorphs that are extensively employed in identifications include vesicle shape, stipe extension length and macroconidial septation and dimensions The morphological characteristics of the teleomorph that is important for identifications are

ascospore septation and dimensions, ascospore number within the asci and perithecial color

Figure 3.4 Microscope and camera use to describe morphological characteristics 3.6 Method of describing the biological characteristics of fungi:

The effects of different nutrient medium, levels of temperature, moisture to the development of fungi, determine the optimum temperature, moisture level for growing Determine the optimum media for culture The growth researching of the mycelium is conducted with 26 strains which have pathogenic ability All experiments were repeated 2 times and getting the average diameter values represents experiments

3.6.1 Growth of the mycelium on different nutrient media:

Firstly, preparing 3 kinds of media, including PDA, OSA, MEA (formula for 1 liter):

 PDA nutrient medium: 200g potatoes minced in 1L water Boiling in 30 minutes, filter the water Then add enough water to 1 liter of water Thereafter, add 20g glucose and 18g agars

 OSA nutrient medium: 30g oatmeal put 1L water Boiling it in 1hour, filter the water Then add enough water to 1 liter of water Thereafter, add 20g glucose and 18g agars

 MEA nutrient medium: Add 20g malt extract and 18g agar in 1L water

Shake well all solutions to dissolve, steamed for 20 minutes at 121°C Lastly, when temperature of solution is about 50°C, pouring them into petri dishes

Secondly, the fungus was transferred into the middle of the petri dish Each strain implanted in 3 types of medium After one day, 3days and 5 days- measure the diameter of the mycelium growing on the medium once (measured perpendicular two-way and take the mean)

Figure 3.5 Three types of nutrient media

a PDA nutrient medium b.OSA nutrient medium c MEA nutrient medium

3.6.2 Growth of the mycelium on PDA nutrient medium at different temperature scales:

Fungus implanted into the middle of the dish containing PDA, this manipulation has to do

in put the box into the incubator cages with different temperature range 50C± 1; 150C±1; 250C ± 1; 350C ±1 After one day, 3days and 5 days measure the diameter of the mycelium growing on the medium once (measured perpendicular two-way and take the mean)

Figure 3.6 Fungus culturing

3.6.3 Growth of the mycelium on PDA nutrient medium at different moisture scales:

The experiment was conducted according to the method of Booth.C.P 1971 NaCl solution

is mixed with different concentrations in the desiccators make us get the moisture as follow:

Table 3.2 Formula to create moisture environment

Put mixed solutions into large desiccators Put the lid on and put them in the laboratory, in the temperature about 23-27°C After 2 days, in different desiccators will have different moisture, depend on NaCl concentration When NaCl concentration is large, moisture of environment is small And vice versa, if the concentration of NaCl is smaller, the environmental moisture will increase PDA nutrient after sterilize autoclaving is poured into sterile box with a thick layer 2 - 3 mm, implanted fungus into the middle of the petri dishes containing the PDA, each desiccators put dishes

Figure 3.7 Moisture experiment

After one day, 3 days, and 5 days- measure the diameter in two dimensions perpendicular and take the average value

4 RESULTS 4.1 Result of sample collection

After collecting soil from 3 locations, 72 soil samples were collected

Table 4.1 List of soil samples is collected