Taxonomy and biology of calonectria in forest soil of vietnam

ACKNOWLEDGEMENTS In order to complete this study course, with the consent of the Department of Forest Resources and Environment Management (FREM), I conduct to 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 Sincerely! Hanoi, September- 2016 Nguyen Thi Loan 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 INTRODUCTION 1.1 Imperativeness 1.2 Study site 1.3 Object of research 1.4 Literature reviews GOALS AND (SPECIFIC) OBJECTIVES 2.1 Goals 2.2 Specific objectives METHODS 3.1 Sampling methods: 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 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 DISCUSSION 37 CONCLUSION 39 REFERENCES LIST OF FIGURES Figure 1.1 Study site Figure 3.1 Soil samples 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 days 33 Figure 4.16 Growth of some mycelium in different moisture after days 34 Figure 4.17 Growth of some mycelium in different moisture after days 35 Figure 4.18 Growth of mycelium in different moisture scales 36 LIST OF TABLES Table 1.1 Calonectria species reported in Vietnam 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 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, 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 communes of Sapa district (Lao Chai, Ta Van, San Sa Ho, Ban Ho) in Lao Cai province and 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 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 There is 21,900 of specially used land and 5700 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 (39%) The average annual temperature is 26°C, ranges from 24.3°C to 28°C December has lowest average monthly Figure4.10 Vesicle and spore of Ca pentaseptata Ca pentaseptata belongs to sphaeropedunculate group Spore size 38.2 (µm) with or septa 4.5.3 Morphological characteristics of Ca illicicola (LT DN 2.1 & LT DN2.2) Figure4.11 Vesicle and spore of Ca illicicola Ca illicicola belongs to obpyriform group Spore size of LT DN2.1 is 37.5 (µm), LT DN2.2 is 36.1 (µm) with or septa 4.6 Biological characteristics of fungi 4.6.1 Growth of the mycelium on different nutrient media Fungal culture experiments in the different nutrient media: PDA nutrient medium; OSA nutrient medium; MEA nutrient medium This experiment is conducted with 26 strains which have pathogenic ability in pathogenicity test (Table 4.4) Growth of the mycelium of 26 fungi, after implementation, the results showed that the growth of fungi in the different nutrient media is very clear 29 a mm 8.00 Growth of mycelium in different nutrient meadiums after days 6.00 OSA 4.00 PDA 2.00 0.00 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 b mm 7.00 Strain Growth of mycelium in different nutrient mediums after days 6.00 5.00 OSA PDA MEA 4.00 3.00 2.00 1.00 0.00 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Strain Figure 4.12 Growth of the mycelium on different nutrient media a after days b after days Above figure shows that OSA nutrient medium is the best for growth of Calonectria Mycelium grows very fast in this medium In types of other medium, they can grow but lower than in OSA medium Moreover, in PDA and MEA nutrient medium, each strain fungus has different growth Some strains grow fast in PDA nutrient medium; on the contrary, some strains grow fast in MEA nutrient medium 30 a b c Figure 4.13 Difference growth of mycelium in OSA, PDA, and MEA nutrient medium a RTN BP3.5.2 b BB BD1.1 c DP BP5.4 In figure 4.13a, sample RTN BP3.5.2 grew fastest in OSA and showed similar growth rate in both other nutrient media In figure 4.13b, sample BB BD1.1 grew fastest in OSA, grew lower in PDA, and grewlowest in MEA In figure 4.13c, sample DP BP5.4 also grew fastest in OSA, but it is opposite to sample BB BD1.1 at other nutrient media Figure 4.13 not only show the different in growth diameter but also show the different in color of fungi Can easy to see diverse color of them in each nutrient medium After this experiment, one thing is 31 showed that, OSA is optimal nutrient medium for growth of Calonectria (figure 4.12, figure 4.13) 4.6.2 Growth of the mycelium on PDA nutrient medium at different temperature scales: Fungal culture experiments in the ladder of different air temperatures: 5°C ± 1; 15°C ± 1; 25°C ± 1; 35°C ± This experiment is conducted with 26 strains which have pathogenic ability in pathogenicity test (Table 4.4) Growth of the mycelium of 26 fungi, after implementation, the results showed that the growth of fungi in the different temperature scale is very clear At a temperature of ≤ 5°C ± and ≥ 35°C ± almost the mycelium of 26 samples could not grow and develop Mycelium systems are worst growth at 15°C ± At 25°C ± 1, fungi grow best and achieve maximum levels depending on the strain of the fungus a b Figure 4.14 Growth of some mycelium in different temperatures a DP BP5.3.2 b.LT DN2.2 The growth data of mycelium at different temperature are followed after days, days However, the difference in the third day is clearer We can see in figure 4.15, at 5°C, and 35°C, almost mycelium systems not grow or grow insignificantly At 15°C, mycelium systems slow grow At 25°C, mycelium systems fastest grow Therefore, optimal temperature for growth of Calonectria is about 25°C 32 Growth of mycelium at different temperature scales after days mm 9.00 8.00 7.00 5°C 6.00 5.00 15°C 4.00 25°C 3.00 35°C 2.00 1.00 0.00 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Strain Figure 4.15 Growth of mycelium at different temperature scales after days 4.6.3 Growth of the mycelium on PDA nutrient medium at different moisture scales Humidity is an important factor that affects the growth of fungi Having 26 strains of fungi were chosen and conducted in this experiment (Table4.4) Most of them are strains which have high ability in pathogenicity (> 20%) The experiment was conducted at different moisture scales: 60%, 70%, 80%, 90% and 100% Research findings of the effects of humidity on the growth of fungi growing through growth diameter of the mycelium system have the following results: All 26 samples Calonectria sp., the mycelium growth is different when they were cultured in various humidity scales The mycelium can grow in air humidity of about 60% -100% However, the growth achieved optimum results at the appropriate moisture; and, growth is slower in unsuitable moisture Growth diameter of the mycelium also depends on each strain that has the largest size at different moisture scales 33 a b c Figure 4.16 Growth of some mycelium in different moisture after days a.LT DN2.1 b.S16 c.RTN BP3.2 Through figure 4.16, we can see that difference growth of mycelium is not clear Even by normal eyes, we could not determine what strain fastest grow or what strain lowest grow However, we can see different growth between different strains Three strains (LT DN2.1, S16, and RTN BP3.2) in above figure is a sample We can easy to see that growth of each is different in the same conditions RTN BP3.2 is fastest grow, S16 is lower grow, and LT DN2 is lowest grow 34 a b c d Figure 4.17 Growth of some mycelium in different moisture after days a.RTN BP3.3 b DP BP5.4 c BB BD1.2 d.RTN BP3.6 After days, difference is more clearly Moisture experiment showed that growth of mycelium does not depend on moisture so much Moreover, each strain has own optimal moisture In figure 4.17a, mycelium of RTN BP3.3 well grow at 60%, 70%, 80%, and low grow at 90%, 100% of moisture On the other hand, in figure 4.17b, mycelium of DP BP5.4 well grow at 90%, 100%, and low grow at 60%, 70%, and 80% of moisture In figure 4.17c, there is no big difference about growth diameter but the color of this strain has a clear difference (at 80%, 90%, 100% mycelium are raised color) Lastly, there is no difference in both color and growth diameter of mycelium (figure 4.17d) It is mean that RTN BP3.6 can well growth in all of moisture scales in this experiment 35 mm 30.00 Growth of mycelium at different moisture scales after days 25.00 100% 20.00 90% 15.00 80% 10.00 70% 5.00 60% 0.00 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 mm Strain Growth of mycelium at different moisture scales after days 30.00 25.00 100% 20.00 90% 15.00 80% 10.00 70% 5.00 60% 0.00 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Strain Figure 4.18 Growth of mycelium in different moisture scales a after days b after days Figure 4.18 showed that the growth of 26 fungi strains is not equal, and they can grow at all of moisture scales: 60%, 70%, 80%, 90%, and 100% Stacked column chart are used to show this thing Each color presents for growing of mycelium at one moisture scale At both of above figure, the difference is various Each strain has own optimal humidity The disparity is not large, but can see that with each sample, growth is different at each scale of moisture 36 DISCUSSION Through internship, and research on Calonectria, I noticed that this is a new research project in the country Therefore, we need to have additional studies and tests on a wider scope I think that it is useful for forestry After research, species have high pathogenic ability on Eucalyptus which were defined including: Ca follicola, Ca pentaseptata, and Ca illicicola In the past: Ca follicola was found on E urophylla × E grandis clone leaf in Guangxi, China by L Lombard et al (2015).Ca pentaseptata was also found with leaf blight symptoms on Eucalyptus in Vietnam by L Lombard, M.J Wingf., P.Q Thu & Crous (2012) Ca illicicola has already been recorded as causing leaf spot on Antilles cherry in Brazil (Silva et al 2001) 41 strains were found, in which only having 26 pathogenic strains This means that not all Calonectria sp are harmful 30 strains were found in the South of Vietnam Most of them belongs vesicle group with various shape of vesicle 11 strains were found in the North of Vietnam Most of them belongs group without vesicle Therefore, Calonectria sp grow better and various in the South of Vietnam One of the main problems in this research is that taxonomy Nowadays, with development of technology, DNA technology withPCR amplification and sequencing is the best choice to taxonomy The shortcoming in here is this method still is limited in Vietnam If we want to appraise fungi samples, we will have to send them to foreign countries such as Korea, with high cost Therefore, I hope that DNA technology will be more developed in Vietnam Secondly, the biological characteristic also is important part In this research, we collected many significant data about growth of Calonectria in different nutrient medium, temperature, and humidity With these inoculation tests, we can define biological 37 characteristics of fungi strains We can know the optimal nutrient medium, temperature, and humidity for growth of them In general, there are characteristics clearly such as nutrient medium or temperature We can find common medium, temperature for optimal growth of all strains On the other hand, it is difficult to find common moisture for optimal growth of them The growth is various with each strain The shortcoming in here is the experiments study on the growth characteristics of Calonectria only done times due to time limitations Therefore, the assessment of the development and growth of the medium types, temperature range, and humidity also mean reference only Should continue to implement and follow up several times to more meaningful results All things considered, there are still many shortcomings need to make better However, it is hoped that this research will have small contribution in order to protect forest plant in future 38 CONCLUSION After research completing, 72 soil samples which were collected in locations: Hoang Lien National Park, Tam Dao National Park, and Binh Phuoc Province Results of isolation showed that 41 strains of Calonectria were found and all of them grow well in culture medium This mean that Calonectria is diversified existence in forest soils of Vietnam Therefore, learning and researching about this fungus are very significant in order to protect diversity and development of flora Conducting to pathogenic with 41 strains, results showed that 15 strains from this not have pathogenic ability, and 26 other strains have the pathogenic ability Experiment on Eucalyptus leaves can show this thing They make leaves become dry with levels depending on each strain In 26 strains, strains have highest pathogenic ability (> 40%); 14 strains have median pathogenic ability (from 20- 40 %); and strains have smaller pathogenic ability (< 20%) These results showed that, Calonectria is a fungus that is threatening to the growth and development of flora Therefore, we should pay attention to learn, to prevent their pathogenic ability In pure culture condition after isolation, 41 strains can grow and develop From that, by using microscope, morphological characteristics of them were fully described (vesicle, spore shape, spore size, and septa) They are put on groups: without vesicle, vesicle withsphaeropedunculate, vesicle with obpyriform, and vesicle with ellipsoid with papillate apex By using DNA technique (PCR amplification and sequencing), fungi strains which highest pathogenic ability are defined: DMCTN2.1 is Ca follicola, RTNBP3.3 is Ca pentaseptata, LTDN2.1 and LT DN22.2 are Ca illicicola 39 By conducting culture experiments in the ladder of different nutrient medium, air temperatures, and moistures, biological characteristics of strains were defined The optimal nutrient medium, the optimal temperature, and the optimal humidity were showed - The moisture experiment completed with types of medium (PDA, OSA, MEA) Although 26 samples can grow in all types of nutrient mediums, OSA still is optimal nutrient medium for growth of Calonectria - In terms of temperature, the results show that the growth of fungi in the different temperature scale is very clear At temperatures less than 5°C, and greater than 35°C, the fungus could not grow or grow but very poorly for some strains At 25°C mushrooms grow very well, that is optimal temperature for growth of Calonectria Lastly, mushrooms are poorly developed at 15°C - Research findings of the effects of humidity on the growth of fungi growing through growth diameter of the mycelium system on 26 strains of Calonectria showed that air humidity in the range of 60% -100%, they are able to grow and develop Furthermore, results also proclaim that the development of the fungus in the aforementioned moisture levels have no higher deviation 40 REFERENCES Booth C, Gibson IAS (1973) Cylindrocladium scoparium CMI Descriptions of Pathogenic Fungi and Bacteria No 362 Booth TH, Jovanovic T, Old KM, Dubzinski MJ (2000) Climatic mapping to identify high – risk areas for Cylindrocladium 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