INTRODUCTION
Imperativeness
Cylindrocladium quinqueseptatum, an anamorph of Calonectria species, was first identified in Vietnam as a fungal pathogen affecting Eucalyptus spp from Hue to the South Calonectria pentaseptata was described by Pham Quang Thu and colleagues in 2012 and has since been found in various regions across Vietnam This research aims to explore the diversity and characteristics of these fungi.
Calonectria Hope that this study will be useful with disease management in the future in order to protect flora.
Study site
Vietnam, situated on the Indochina Peninsula in Southeast Asia, shares borders with China to the north, Cambodia and Laos to the west, and the Gulf of Thailand to the southwest, while the East Sea lies to the east and south Covering an area of 331,698 km², three-quarters of Vietnam's landscape is hilly, with 327,480 km² of terrestrial land and 4,500 km² of inland sea The coastal location influences the climate, resulting in an average relative humidity of 84% year-round, annual precipitation ranging from 1,200 to 3,000 mm, and sunshine hours between 1,500 and 3,000 Temperatures in Vietnam can vary significantly, ranging from 5°C to 37°C.
Vietnam's diverse terrain and climate create an ideal environment for a wide variety of flora and fauna, particularly in its forests The predominantly hilly landscape underscores the significance of forests in the country However, the hot and humid climate, combined with heavy rainfall in certain regions, fosters the growth of pests and harmful fungi This study focuses on three key locations with distinct topographic and climatic characteristics: Hoang Lien National Park, Tam Dao National Park, and Binh Phuoc province.
Hoang Lien National Park, located in the Hoang Lien Son Range, spans 28,509 hectares across four communes in Sapa district and two in Tan Uyen district, straddling Lao Cai and Lai Chau provinces This unique park features a blend of subtropical and alpine climates, characterized by rugged terrain and peaks exceeding 1,000 meters, including Fansipan at 3,143 meters Renowned for its rich biodiversity, Hoang Lien is home to over 2,000 plant species, including notable varieties like the Bo tree, azaleas, and plums, making it one of Vietnam's most significant centers for flora diversity.
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
Tam Dao National Park, located approximately 85km northwest of Hanoi, is a protected area in North Vietnam that spans 80 km from northwest to southeast It features over 20 peaks, including the highest summit, Tam Dao North, at 1,592m The park's unique topography is characterized by sharp peaks and deep partitions, resulting in diverse climatic conditions and varied rainfall Tam Dao is home to eight forest types, including tropical moist evergreen and bamboo forests, with over 2,000 plant species, of which 904 are beneficial to humans The park also boasts rich biodiversity, housing 840 animal species, including 11 endemic species, and 22 species endemic to North Vietnam.
Binh Phuoc Province, located in the Southeast region of Vietnam and north of Ho Chi Minh City, shares a border with Cambodia The province features relatively flat terrain with elevations ranging from 50m to 200m, increasing to approximately 500m near the Dak Nong border, and the highest point is Ba Ra Mountain at 736m Covering 337,000 ha, forestry land constitutes 49% of the province's total area, primarily found in the northeast and southeast regions, while the remaining land is utilized for perennial cash crops across a total agricultural area of 293,700 ha The province experiences an average annual rainfall of 2,280 mm, with variations between 1,900 mm and 2,731 mm, and rainfall distribution is uneven, increasing from Southeast to Northwest The average annual temperature is around 26°C, with December being the coolest month at 24.3°C and April the warmest at 28°C.
Object of research
Flora plays a crucial role in forest ecosystems, reflecting the genetic biodiversity of forest resources and influencing forest diversity, scientific value, landscape aesthetics, and economic benefits While certain locations provide optimal conditions for flora growth, factors such as high temperatures and heavy rainfall can also promote the development of pests and diseases, including the pathogenic fungus Calonectria This fungus poses a significant threat to plant health, making its management essential for the protection and growth of trees in these ecosystems.
Most of Calonectria species are readily recovered from soil (Crous, 2002) Therefore, this study also conducts isolate them from soil samples.
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
In 1979, Rossman reclassified Ca daldiniana as a synonym of Ca pyrochroa This nectrioid fungus is characterized by its vividly colored ascocarp wall structure, which transforms to a blood-red hue when exposed to a 3% KOH solution, exhibiting a warty to scaly texture.
Cylindrocladium (Cy.) anamorph is often difficult to identify at the species level due to the limited morphological features of its teleomorph As a result, specimens can typically only be classified accurately when the anamorph is present, highlighting the importance of this form in identification processes.
The anamorph genus Cylindrocladium, first described by Morgan in 1892 in the U.S.A on Gleditsia triacanthos, is characterized by branched conidiophores that produce cylindrical conidia Despite Morgan's omission of the unique stipe extension ending in a distinctive vesicle, the genus has since been recognized for its widespread distribution in subtropical and tropical regions, with various species identified as pathogens affecting numerous plants (Crous, 2002; L Lombard et al., 2010).
Lombard's research indicates that Calonectria is part of the Nectriaceae family within the Hypocreales order, which has undergone extensive review The Nectriaceae is characterized by its uniloculate ascomata, which range in color from orange to purple and are not embedded in well-developed stromata This family comprises around 20 genera that hold significant socio-economic importance, with Calonectria being distinctly recognized among them.
Cylindrocladium anamorphs and relevance as plant pathogens
Initially considered saprobes, Calonectria fungi were first proven pathogenic by Massey in 1917 and later by Anderson in 1919 with Ca morganii (formerly Cy scoparium) Since then, Calonectria species have been linked to various disease symptoms across numerous hosts globally, affecting plants in around 30 different families, as noted by several researchers including Booth & Gibson (1973) and French & Menge.
In 1978, research indicated that the number of plant families affected by pathogens is closer to 100, with around 335 plant host species identified These hosts encompass significant forestry, agricultural, and horticultural crops, suggesting that the impact of these plant pathogens may have been underestimated (L Lombard et al., 2010).
Calonectria species are primarily linked to disease reports in forestry, affecting hosts from five plant families, notably Fabaceae (Acacia spp.), Myrtaceae (Eucalyptus spp.), and Pinaceae (Pinus spp.) The diseases manifest through various symptoms, including leaf spots, stem lesions, cankers on stems or crowns, as well as root rot, crown rot, and stem rot In Vietnam, these issues are particularly relevant.
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
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
Ca pentaseptata Leaf blight Eucalyptus hybrid Crouset al 2012
This study, titled "Research on Taxonomy and Biology of Calonectria in Forest Soil of Vietnam," focuses on isolating and identifying fungal species within the genus Calonectria using DNA sequence analysis and morphological characteristics The research aims to explore the biological traits and pathogenicity of these isolates Ultimately, the findings are expected to contribute to effective plant disease management and the conservation and enhancement of floral biodiversity in Vietnam.
GOALS AND (SPECIFIC) OBJECTIVES
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 of fungal strains.
Specific objectives
Soil samples were collected from Hoang Lien National Park, Tam Dao National Park, and Binh Phuoc province to isolate fungal species from the genus Calonectria DNA extraction and identification of the isolates were performed through DNA sequence analysis Additionally, the morphological characteristics of the spores were examined using an Olympus BX 50 microscope.
- Determine the biological characteristics of fungal strains the pathogenicity of isolation by inoculation tests.
METHODS
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 have been identified as causing leaf spot symptoms on Eucalyptus species (Crous P.W et al, 2002) In response, we collected soil samples from various Eucalyptus plantations, focusing on locations with moist soil conditions that are favorable for the growth of Calonectria species.
Soil samples will be collected from areas with symptomatic plants, as well as from natural forest soil These samples will be stored in labeled plastic bags, indicating the time and location of collection, and then transported to the laboratory for analysis.
Isolation methods
Soil collected cup: can use the urine specimen collection cups or some kinds of cup to hold food
Sterilized alfalfa seeds : take the alfalfa seeds soak into the bleach solution (2.5%), about 30 seconds later, and then rewash by distilled water 5 to 6 times
To prepare a soil sample for analysis, start by thoroughly blending the soil Next, remove any roots, leaves, or other debris from the sample, and crush any large soil clumps into finer particles for better consistency.
Spray bottle of distilled water
To prepare PDA nutrient medium, start by mincing 200g of potatoes and boiling them in 1 liter of water for 30 minutes After boiling, filter the mixture and adjust the volume back to 1 liter with water Next, incorporate 20g of glucose and 18g of agar, stirring thoroughly to ensure complete dissolution Steam the mixture at 121°C for 20 minutes, then allow it to cool to approximately 50°C before pouring it into petri dishes.
To prepare an antibiotic PDA nutrient medium, dissolve 0.2g of streptomycin in 100ml of distilled water, then mix this solution with a heated blend of potatoes, agar, and glucose at approximately 50°C After thoroughly shaking the mixture, pour it into petri dishes for solidification.
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.
Methods of pathogenicity testing
Pathogenicity test was conducted in leaves of Eucalyptus
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
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
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
In a 25 µl PCR reaction mixture, approximately 20 ng of template DNA was amplified using 1 x PCR reaction buffer, 200 µM each of dATP, dCTP, dGTP, and dTTP, and primer pairs EF1-728F and EF2 to target the translation elongation factor gene region The reaction included 1.25 units of Taq DNA polymerase (Roche Diagnostics GmbH, Mannheim, Germany) and followed a specific thermal cycling protocol: an initial denaturation at 96°C for 5 minutes, 30 cycles of denaturation at 94°C for 30 seconds, annealing at 52°C for 30 seconds, extension at 72°C for 90 seconds, and a final extension at 72°C for 7 minutes, using a GeneAmp PCR System 2700 (PerkinElmer, Norwalk, Connecticut).
Each experiment included a negative control where water replaced the template DNA The PCR products were then analyzed by electrophoresis at 75 V for 1 hour on a 0.8% (w/v) agarose gel using a 0.5 x TAE buffer, which contains 0.4 M Tris, 0.05 M NaAc, and 0.01 M EDTA at pH 7.85 Visualization of the gel was performed under UV light with a GeneGenius Gel imaging system.
The PCR products were purified using the NucleoSpin Extract 2 in 1 Purification Kit from Macherey-Nagel GmbH, Germany, and analyzed with the Documentation and Analysis System from Syngene, Cambridge, UK, following ethidium bromide staining For the cycle sequencing reaction, 20 to 40 ng of purified PCR products and 10 pmol primers were combined in a total volume of 10 µl, utilizing the ABI PRISM BigDye Terminator v3.0 Cycle Sequencing Ready Reaction Kit from PE Biosystems, Foster City, CA, USA, which includes AmpliTaq DNA Polymerase The reaction conditions involved an initial denaturation at 94°C for 5 minutes, followed by 25 cycles of denaturation at 96°C for 10 seconds and annealing at 55°C.
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.
Method of describing the morphological characteristics of fungi
Morphological and phenotypic characters have historically been crucial in describing fungal species, as outlined by various researchers (Brasier 1997, Taylor et al 2000) and mandated by the ICBN (McNeill et al 2005) However, there has been a significant shift towards utilizing biological and phylogenetic characters for species delimitation, as noted in studies on Calonectria species (Crous et al 2004b, 2006a) To accurately observe and document fungal characteristics, it is essential to examine them microscopically, focusing on traits such as shape, bulkhead, and size, while also measuring spore length and capturing images for further analysis.
Key characteristics for identifying anamorphs include vesicle shape, stipe extension length, and the septation and dimensions of macroconidia For teleomorph identification, crucial morphological traits are the septation and dimensions of ascospores, the number of ascospores within asci, and the color of the perithecia.
Method of describing the biological characteristics of fungi
This study investigates the impact of various nutrient media, temperature levels, and moisture conditions on fungal development, aiming to identify the optimal temperature and moisture for growth Additionally, it seeks to determine the most effective media for culturing fungi Research was conducted on the mycelium growth of 26 pathogenic strains, with all experiments repeated twice to ensure accuracy, yielding average diameter values that represent the findings.
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
The fungus was placed in the center of the petri dish, with each strain cultivated in three different types of medium After one, three, and five days, the diameter of the mycelium growth on the medium was measured twice perpendicularly, and the average was calculated.
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:
Fungi were inoculated into the center of Petri dishes containing Potato Dextrose Agar (PDA) and then placed in incubators set at various temperature ranges: 5°C ± 1, 15°C ± 1, 25°C ± 1, and 35°C ± 1 After 1, 3, and 5 days, the diameter of the mycelium growth on the medium was measured in two perpendicular directions, and the average diameter was calculated.
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
Place mixed solutions in large desiccators and seal them before storing in the laboratory at a temperature of 23-27°C After two days, varying moisture levels will be observed in different desiccators, influenced by the concentration of NaCl; higher NaCl concentrations lead to lower environmental moisture, while lower concentrations result in increased moisture Following autoclaving, pour sterilized PDA nutrient into sterile boxes with a thickness of 2-3 mm, and inoculate the center of the petri dishes with fungus before placing them in the desiccators.
After one day, 3 days, and 5 days- measure the diameter in two dimensions perpendicular and take the average value.
RESULTS
Result of sample collection
After collecting soil from 3 locations, 72 soil samples were collected
Table 4.1 List of soil samples is collected
No Name No Name No Name No Name
Result of isolation, and culturing the fungus
Figure 4.1 Some strains under stereoscope
After isolation and culturing the fungus, 41 samples were isolated
Table 4.2 List of fungi strains were found
No Name No Name No Name No Name
1 BB BD1.1 12 DP BP5.2 22 RTN BP3.3 32 S15
2 BB BD1.2 13 DP BP5.3.1 23 RTN BP3.4 33 S16
3 BB BD1.3 14 DP BP5.3.2 24 RTN BP3.5.1 34 S24
4 BB BD1.4 15 DP BP5.4 25 RTN BP3.5.2 35 S29
5 DMC TN1.1 16 LT DN2.1 26 RTN BP3.6 36 S30.1.1
6 DMC TN1.2 17 LT DN2.2 27 RTN BP3.7 37 S30.1.2
7 DMC TN1.3 18 LT DN3 28 RTN BP5.1 38 S30.1.3
8 DMC TN2.1 19 RTN BP3.1.1 29 RTN BP5.2 39 S30.2
9 DMC TN2.2 20 RTN BP3.1.2 30 RTN BP5.3 40 S36.1
10 DMC TN2.3 21 RTN BP3.2 31 TD1 41 S36.2
This fungus has cylindrical spore with septa are described in figure 4.2
Figure 4.2 Spore of Calonectria Table 4.3 Some characteristics of 41 fungi strains
8 DMCTN2.1 33.1 2 Ellipsoid with papillate apex
9 DMCTN2.2 36.1 1 or 2 Ellipsoid with papillate apex
10 DMCTN2.3 27.4 1 or 2 Ellipsoid with papillate apex
13 DPBP5.3.1 40.3 1 or 3 Ellipsoid with papillate apex
31 S15 36.1 1 or 2 Ellipsoid with papillate apex
32 S16 38.2 1 or 2 Ellipsoid with papillate apex
41 TD1 32 2 or 3 Ellipsoid with papillate apex These tables show results of some characteristics of fungi In there, can divide them in
The study identifies four distinct groups based on vesicle characteristics and spore size The largest group comprises 14 strains without vesicles, featuring spores ranging from 11 to 15 µm with 1 to 5 septa The second group, consisting of 14 strains with sphaeropedunculate vesicles, has spores sized between 29.3 and 46.7 µm and 2 to 3 septa A smaller group contains 7 strains with ellipsoid vesicles and papillate apices, displaying spore sizes from 27.4 to 40.3 µm and 1 to 3 septa The smallest group includes 6 strains characterized by obpyriform vesicles, with spore sizes ranging from 34.7 to 37.5 µm and 1 to 3 septa.
Figure 4.3 Four groups of isolates a Sphaeropedunculatevesicle b Obpyriform vesicle c.Ellipsoid vesicle with papillate apex d Without vesicle
Fungi are classified into two primary groups: those with vesicles and those without The vesicle group is further divided into three subcategories based on vesicle shape: sphaeropedunculate, obpyriform, and ellipsoid with papillate apex This classification helps clarify the distinct characteristics of fungi.
Pathogenicity testing
Pathogenicity testing was conducted with 41 isolated strains (Table 4.2) As we can see, fungal isolates were found which have pathogenic ability in trees In this case is leaf of
Figure 4.4 Pathogenic ability of fungal strains
Depend on above figure, pathogenic ability of fungi strains was found:
• 15 strains of fungi don’t have pathogen ability
• 3 strains of fungi have low pathogen ability
• 14 strains of fungi have medium pathogen ability
• 9 strains of fungi have high pathogen ability
From that, fungi strains have high ability in pathogenicity were identified We can define pathogenic strains and pathogenic ability of them
Figure 4.5 The process of pathogenicity of Calonectria on Eucalyptus leaf a first day b third day c fifth day
Table 4.4 presents 26 pathogenic strains along with their respective pathogenic levels, highlighting the varying abilities of each strain Notably, the strain with the highest pathogenicity is RTN BP3.3, which exhibits a pathogenicity level of 0.64.
Table 4.4 Pathogenic strains and pathogenic levels of them
Pathogenic level No Sample Name Pathogenic level
Taxonomy by DNA technique: PCR amplification and sequencing
The EF1-728F and EF2 primer pairs were utilized to amplify a fragment of the translation elongation factor gene region Phylogenetic trees for this gene region were constructed using the Maximum Likelihood (ML) algorithm.
After result of pathogenicity testing, 9 strains have high pathogen ability
However, time is limited, so choosing 4 strains in 9 strains which have highest pathogenicity to expertise: DMC TN2.1, RTN BP3.3, LT DN2.1, and LT DN2.1
The article lists various strains of fungi, including *Ca cylindrospora* (CBS119669, CBS110666, GQ421796, FJ918557), *Ca insularis* (CBS114559, CBS114558, FJ918555, FJ918556), and *Ca variabilis* (CBS114677, CBS112691, GQ267334, GQ267335) It also mentions *Ca pseudohodgesii* (CBS134813, CBS134818, KM395815, KM395817), *Ca brasiliensis* (CBS230.51, CBS114257, GQ267328, GQ267329), and *Ca hodgesii* (CBS133609, CBS133608, KC491225, KC491224) Additionally, the strains *Ca sulawesiensis* (CMW14878, CMW14857, GQ267340, GQ267343) and *Ca foliicola* (CMW31394, CBS136641, KJ462801, KJ462800) are included, highlighting the diversity of fungal species cataloged in this research.
CBS134812 Ca maranhensis KM395862 CBS134811 Ca maranhensis KM395861 CBS136643 Ca terrestris KJ462892
CBS136642 Ca terrestris KJ462891 CBS136096 Ca papillata KJ462848 CBS136084 Ca papillata KJ462847
CBS123695 Ca cerciana FJ918560 CBS123693 Ca cerciana FJ918559
CBS134823 Ca pseudocerciana KM395874 CBS134822 Ca pseudocerciana KM395873 CBS134815 Ca propaginicola KM395866 CBS134816 Ca propaginicola KM395867
Figure 4.6 Classification tree of DMC TN2.1
Through figure 4.6, concluding that DMCTN2.1 is Ca follicola
CBS136087 Ca pentaseptata KJ462853 CBS133349 Ca pentaseptata JX855958
CBS112144 Ca reteaudii FJ918537 CBS112143 Ca reteaudii FJ918536 CBS123696 Ca pseudoreteaudii FJ918542 CBS123694 Ca pseudoreteaudii FJ918541 CBS136634 Ca microconidialis KJ462843 CBS136633 Ca microconidialis KJ462842
CBS112146 Ca queenslandica FJ918543 CBS112151 Ca terrae reginae FJ918545
CBS112634 Ca terrae reginae FJ918546 CBS112155 Ca queenslandica FJ918544
CBS114813 Ca acicola GQ267292 CBS114812 Ca acicola GQ267291 CBS112954 Ca australiensis GQ267293 copy CBS112954 Ca australiensis GQ267293 CMW27253 Ca crousiana HQ285823
The article lists various fungal species along with their corresponding accession numbers Notable entries include Ca angustatum (CBS112133, FJ918552) and Ca angustata (CBS114544, FJ918551) Additionally, Ca hurae is represented by two entries (CBS114551, FJ918548), while Ca rumohrae appears twice (CBS111431, FJ918549 and CBS114529, FJ918550) Ca hawksworthii is also mentioned with two identical entries (CBS111870, FJ918558), as is Ca leguminum (CBS728.68, FJ918547) Lastly, Ca multiseptata is recorded twice as well (CBS112682, FJ918535).
Figure 4.7 Classification tree of RTN BP3.3
Through figure 4.7, concluding thatRTNBP3.3 is Ca pentaseptata
CBS136249 Ca magnispora KJ462841 copy CBS136249 Ca magnispora KJ462841
IMI35428 Ca pacifica AY725724 CBS114038 Ca pacifica GQ267320
CBS116159 Ca curvispora GQ267302 CBS116159 Ca curvispora GQ267302 copy
CBS114073 Ca asiatica AY725705 CBS112711 Ca asiatica AY725702
CBS112221 Ca colombiensis AY725712 CBS112220 Ca colombiensis AY725711 CBS413.67 Ca kyotensis GQ267307
CBS113783 Ca ilicicola AY725729 CBS190.50 Ca ilicicola AY725726
CBS112934 Ca sumatrensis AY725735 CBS112829 Ca sumatrensis AY725733 CBS136081 Ca sphaeropedunculata KJ462890 copy
CBS136081 Ca sphaeropedunculata KJ462890 CBS136091 Ca aconidialis KJ462786 CBS136086 Ca aconidialis KJ462785
The article lists several fungal species along with their respective identifiers, including Ca parakyotensis (CBS136095, KJ462852), Ca expansa (CBS136078, KJ462797; CBS136247, KJ462798), Ca pseudokyotensis (CMW31439, KJ462881), Ca pluriramosa (CBS136976, KJ462882), Ca arbusta (CPC23481, KJ462789; CBS136079, KJ462787), and Ca guangxiensis (CBS136094, KJ462804).
Figure 4.8 Classification trees of LT DN2.1, and LT DN2.2
Through figure 4.8, concluding that LT DN2.1, and LT DN2.2 are Ca illicicola.
Morphological characteristics of fungi
This study aims to define the morphological characteristics of fungi by using a microscope to observe, describe, and measure spore sizes Additionally, three species identified through DNA taxonomy are detailed in terms of their morphological features.
4.5.1 Morphological characteristics of Ca follicola (DMC TN2.1)
Figure4.9 Vesicle and spore of Ca follicola
Ca follicola belongs to ellipsoid with papillate group
Spore size is 33.1 (àm) with 2 septa
4.5.2 Morphological characteristics of Ca pentaseptata (RTN BPP3.3)
Figure4.10 Vesicle and spore of Ca pentaseptata
Ca pentaseptata belongs to sphaeropedunculate group
Spore size 38.2 (àm) with 2 or 3 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 (àm), LT DN2.2 is 36.1 (àm) with 1 or 2 septa.
Biological characteristics of fungi
4.6.1 Growth of the mycelium on different nutrient media
Fungal culture experiments were conducted using various nutrient media, including PDA, OSA, and MEA, to assess the growth of mycelium from 26 pathogenic strains The results of the pathogenicity test are detailed in Table 4.4, highlighting the effectiveness of each medium in supporting fungal growth.
26 fungi, after implementation, the results showed that the growth of fungi in the different nutrient media is very clear
Figure 4.12 Growth of the mycelium on different nutrient media a after 3 days b after 5 days
The OSA nutrient medium is identified as the optimal environment for the growth of Calonectria, facilitating rapid mycelium development While growth is also observed in PDA and MEA nutrient media, it is notably less vigorous compared to OSA Additionally, the growth rates of different fungal strains vary significantly between PDA and MEA, with some strains thriving in PDA while others exhibit faster growth in MEA.
Growth of mycelium in different nutrient meadiums after 3 days
Growth of mycelium in different nutrient mediums after 5 days
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 the experiments depicted in Figure 4.13, various samples exhibited distinct growth patterns across different nutrient media Sample RTN BP3.5.2 demonstrated the fastest growth in OSA, with comparable rates in other media Conversely, sample BB BD1.1 thrived in OSA but showed reduced growth in PDA and the least in MEA Similarly, sample DP BP5.4 also grew best in OSA, contrasting with BB BD1.1's performance in other media The figures not only illustrate variations in growth diameter but also highlight the diverse colors of fungi across the nutrient media Overall, the results indicate that OSA serves as the optimal nutrient medium for the growth of Calonectria.
4.6.2 Growth of the mycelium on PDA nutrient medium at different temperature scales:
Fungal culture experiments were conducted at varying air temperatures of 5°C ± 1, 15°C ± 1, 25°C ± 1, and 35°C ± 1, utilizing 26 pathogenic strains The results indicated a significant impact of temperature on mycelial growth, with minimal development observed at temperatures ≤ 5°C ± 1 and ≥ 35°C ± 1 Notably, mycelium growth was poorest at 15°C ± 1, while optimal growth occurred at 25°C ± 1, achieving maximum levels depending on the specific fungal strain.
Figure 4.14 Growth of some mycelium in different temperatures a DP BP5.3.2 b.LT DN2.2
Mycelium growth was monitored over 3 and 5 days, with significant differences observed by the third day As illustrated in Figure 4.15, mycelium systems showed minimal growth at 5°C and 35°C At 15°C, growth was slow, while the most rapid growth occurred at 25°C, indicating that this temperature is optimal for mycelium development.
Figure 4.15 Growth of mycelium at different temperature scales after 3 days
4.6.3 Growth of the mycelium on PDA nutrient medium at different moisture scales
Humidity significantly influences fungal growth, as demonstrated by an experiment involving 26 selected strains of fungi, predominantly with high pathogenicity rates exceeding 20% The study was conducted across five distinct moisture levels: 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 exhibits varying mycelium growth rates depending on humidity levels, thriving in air humidity ranging from 60% to 100% Optimal growth occurs at specific moisture levels, while unsuitable humidity results in slower growth Additionally, the growth diameter of the mycelium varies by strain, with each strain achieving its largest size at different moisture scales.
Growth of mycelium at different temperature scales after 3 days
Figure 4.16 Growth of some mycelium in different moisture after 3 days a.LT DN2.1 b.S16 c.RTN BP3.2
Figure 4.16 illustrates that the growth rates of mycelium strains are not immediately discernible to the naked eye; however, notable differences exist among various strains For instance, the strains LT DN2.1, S16, and RTN BP3.2 demonstrate distinct growth patterns under identical conditions Specifically, RTN BP3.2 exhibits the fastest growth rate, while S16 shows moderate growth, and LT DN2.1 has the slowest growth rate.
Figure 4.17 Growth of some mycelium in different moisture after 5 days a.RTN BP3.3 b DP BP5.4 c BB BD1.2 d.RTN BP3.6
After 5 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 a b c d
Figure 4.18 Growth of mycelium in different moisture scales a after 3 days b after 5 days
Figure 4.18 illustrates the varying growth rates of 26 fungal strains across different moisture levels: 60%, 70%, 80%, 90%, and 100% The stacked column chart effectively highlights these differences, with each color representing the growth of mycelium at a specific moisture level While the disparities in growth are not significant, it is evident that each strain has its own optimal humidity, leading to distinct growth patterns at each moisture scale.
Growth of mycelium at different moisture scales after 3 days
Growth of mycelium at different moisture scales after 5 days
DISCUSSION
During my internship and research on Calonectria, I discovered that this represents a novel research initiative in the country Consequently, there is a necessity for expanded studies and tests across a broader scope I believe this research will significantly benefit the forestry sector.
Research has identified three highly pathogenic species affecting Eucalyptus: Ca follicola, Ca pentaseptata, and Ca illicicola Ca follicola was first discovered on E urophylla × E grandis clone leaves in Guangxi, China, as reported by L Lombard et al in 2015 Similarly, Ca pentaseptata was observed causing leaf blight symptoms on Eucalyptus in Vietnam, documented by L Lombard, M.J Wingf., P.Q Thu, and Crous in 2012 Additionally, Ca illicicola has been recorded as a cause of leaf spot on Antilles cherry in Brazil, according to Silva et al in 2001.
41 strains were found, in which only having 26 pathogenic strains This means that not all Calonectria sp are harmful
A study identified 30 strains of Calonectria sp in southern Vietnam, predominantly belonging to the vesicle group with diverse vesicle shapes In contrast, only 11 strains were discovered in northern Vietnam, mostly belonging to the non-vesicle group This indicates that Calonectria sp thrives more effectively and exhibits greater diversity in southern Vietnam.
A significant challenge in this research is the current limitations in taxonomy, particularly in Vietnam With advancements in technology, DNA methods such as PCR amplification and sequencing have emerged as the most effective tools for taxonomy However, these techniques are still not widely available in Vietnam, necessitating the costly process of sending fungal samples to foreign countries like Korea for analysis It is my hope that the development of DNA technology will progress further in Vietnam to address these challenges.
The biological characteristics of fungi strains are crucial for understanding their growth patterns This research gathered significant data on the growth of Calonectria across various nutrient media, temperatures, and humidity levels Through inoculation tests, we identified the optimal conditions for each strain, particularly focusing on nutrient medium and temperature, where commonalities emerged However, determining a universal moisture level for optimal growth proved challenging, as growth varied significantly among strains.
The shortcoming in here is the experiments study on the growth characteristics of
Calonectria was assessed only twice due to time constraints, which limits the evaluation of medium types, temperature ranges, and humidity levels to a reference point To achieve more meaningful results, it is essential to continue implementing and conducting follow-up assessments multiple times.
In summary, while there are significant shortcomings that need to be addressed, this research aims to make a valuable contribution toward the future protection of forest plants.
CONCLUSION 39 REFERENCES
After research completing, 72 soil samples which were collected in 3 locations: Hoang Lien National Park, Tam Dao National Park, and Binh Phuoc Province
Isolation results revealed 41 strains of Calonectria thriving in culture medium, indicating the diverse presence of this fungus in Vietnam's forest soils Consequently, studying and researching Calonectria is crucial for preserving biodiversity and promoting the development of flora.
Conducting to pathogenic with 41 strains, results showed that 15 strains from this do not have pathogenic ability, and 26 other strains have the pathogenic ability Experiment on
Eucalyptus leaves exhibit varying levels of dryness based on different strains, with a study revealing that out of 26 strains, 9 demonstrate high pathogenic ability (over 40%), 14 have median pathogenic ability (20-40%), and 3 show low pathogenic ability (under 20%) These findings indicate that Calonectria is a significant fungal threat to plant growth and development, highlighting the need for further research and preventive measures to mitigate its pathogenic effects.
In a controlled pure culture environment, 41 strains were successfully isolated and cultivated Microscopic analysis revealed detailed morphological characteristics, including vesicle types, spore shapes, spore sizes, and septal structures These strains were categorized into four distinct groups: those without vesicles, those with sphaeropedunculate vesicles, those with obpyriform vesicles, and those with ellipsoid vesicles featuring a papillate apex.
Using DNA techniques such as PCR amplification and sequencing, four fungal strains with the highest pathogenic potential have been identified: DMCTN2.1 as Ca follicola, RTNBP3.3 as Ca pentaseptata, and LTDN2.1 and LT DN22.2 as Ca illicicola.
Through a series of culture experiments involving various nutrient media, air temperatures, and moisture levels, the biological characteristics of the strains were analyzed The study identified the optimal nutrient medium, temperature, and humidity conditions for growth.
The moisture experiment conducted with three nutrient mediums—PDA, OSA, and MEA—revealed that while 26 samples can thrive in all three, OSA is the optimal medium for the growth of Calonectria.
Fungal growth is significantly influenced by temperature, with optimal growth occurring at 25°C for Calonectria Growth is severely inhibited at temperatures below 5°C and above 35°C, while development is also poor at 15°C.
Research on the impact of humidity on fungal growth revealed that 26 strains of Calonectria thrive in air humidity levels between 60% and 100% The findings indicate that within this moisture range, the mycelium system exhibits consistent growth without significant variation in development.
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