Trichoderma asperellum: First report in Bangladesh

The experiment was conducted at four laboratory, Bangladesh Agricultural University, Mymensingh to identify T. asperellum and its characterization was obtained from the rhizosphere of 49 different crops in 109 different locations of 36 districts in Bangladesh. Fifteen isolates of 88 isolates in T. asperellum were characterized on morphological and physiological features. All of the isolates were produced at 35 °C, but only TR27 and TR45 were grown and sporulate at 40 °C. Mycelial growth of all the isolates was reduced with the increasing of pH value. Molecular characterization in four isolates of TR27, TR45, TR70 and TR85 were studied at ITS and TEF region nucleotide sequencing among 15 isolates of T. asperellum and the morphological characterization at ITS and tef1 nucleotide sequencing was assimilated with molecular characterization. The isolates of TR45, TR70 and TR85 were found with 98% homology and TR27 exhibited 88% against their respective closest isolate. The isolates of TR27 and TR85 marked their respective nearest homology at 96%, while TR45 showed 99%, and 93% homology was signified with TR70 in TEF sequences. Three isolates (TR45, TR70 and TR85) were identified as T. asperellum of 100% bootstrap value and TR27 isolate was also recognized with 72% bootstrap value in phylogenetic tree. In phylogenetic analysis, tef1 extended better differentiation among the Trichoderma isolates within and among the groups of closely related species than the rDNA of the ITS region in reflecting wider variability in the isolates while a higher transversion ratio and evolutionary divergence were defined. Moreover the exploring of scientific and useful diversification of Trichoderma isolates on physiological and molecular traits has deemed in research fields. IMPORTANCE Trichoderma is an important genus in different aspect of uses as biofertilizer and bioremediation in agriculture. Specific identification in physiological and molecular characterization has also foremost attention with genome sequences as stored in databases. The diversity and genetic distances were ascertained among the isolates in phylogenetic analysis with the sequence data of the ITS region and tef1 of known Trichoderma strains was found in GenBank. Consequently, the databases of Trichoderma asperellum in molecular characterization can be frequently studied in the beneficial scope of agriculture. Trichoderma isolates of Rhizosphere soil in 4 locations with crops of (TR27) Sadar, Moulvibazar (Rice), (TR45) Sadar, Mymensingh (Sweet gourd), (TR70) Chapra, Chapai Nawabganj (Sesame) and (TR85) Nayanpur, Lalmonirhat (Maize) were found to be substantiated as Trichoderma asperellum strain in Bangladesh that were confirmed on physiological and molecular characters, and utilization of this strain can run faster in invasive technologies to control crop diseases.


INTRODUCTION 64
Identification of Trichoderma spp. is challenging in consequential to agricultural beneficial 65 fungi. Pathogens are controlled by biological agents, because they are more natural and 66 environmentally acceptable alternative to existing chemical treatments (1). Application of 67 Trichoderma spp. in biofertilizers and biopesticides is used as bioeffectors (2, 3) and it also 68 functions as bioremediation for heavy metals and other pollutions (4, 5). 69 Trichoderma has significant attributes as biocontrol agents (6) in controlling soil borne plant 70 pathogenic fungi such as Fusarium, Sclerotium and Rhizoctonia (7,8,9). Trichoderma spp. also 71 acts as an antagonistic effects over almost 18 genera and 29 species of pathogenic fungi, and a 72 variety of pathogenic bacteria. Trichoderma spp. also in the biocontrol mechanisms exerts 73 competition and mycoparasitism and often it causes the stimulation in plant resistance and 74 immune system (10). 75 Trichoderma spp. shows mycoparasitism against pathogens in antagonistic mechanism. Firstly, 76 Trichoderma recognizes the pathogen and its mycelium proceeds in straight way with pathogen 77 mycelium in a coiling fashion that results in dissolution with death of the pathogen. As a result, 78 cell wall-degrading enzymes (CWDEs), such as chitinases, glucanase, and proteases are 79 produced due to the mycoparasitism, which penetrating the pathogen mycelium and nourishes its 80 nutrients from dissolved pathogen (11,12,13). Trichoderma spp. also secrets secondary 81 metabolites that induces the pathogen reduce. Different kind of substances as isonitrile, 82 diketopiperazines, sesquiterpenes, stemids, polyketides, alkylpyrones, and peptaibols are 83 produced from various Trichoderma species (14). 84 Several bioagents in 1980 were tested against plant pathogen but Trichoderma and, Gliocladium 85 have gained the maximum success. So, bioagents have tremendous potential effect and can be 86 exploited successfully in modern agriculture for control of plant diseases (15). 87 Effects of temperature on the growth and optimum sporulation of Trichoderma viride and T. 88 harzianum are happened at 30-35 °C (16). Temperature ranges from 20 °C to 37 °C are 89 favorable for best growth and sporulation, and growth and sporulation are also reduced at below 90 20 °C (17). Maximum growth and sporulation of T. viride are influenced between pH 4.5 to 5.5 91 (17). The highest growth and sporulation are also noticed at pH 5-7 (18). Potentiality of 92 Trichoderma species as a bio-agent has been greatly impressed by pH, and the most favorable 93 pH range of 6.5-7.5 is found in the growth and sporulation of Trichoderma (19). defined the Trichoderma isolates as T. virens (11 isolates), T. asperellum (15), T. harzianum 103 (14) and T. longibrachiatum (32) based on the DNA sequence data analysis of the elongation 104 factor 1-alpha gene (tef1). 105 Significant effect of BAU-Biofungicide (Trichoderma based preparation) is observed in 106 controlling the nursery diseases of litchi (24) and BAU-Biofungicide also exerts in inhibiting the 107 leaf blight of wheat (25). BAU-Biofungicide is a unique biocontrol agent that greatly influence 108 in higher germination and plant stand, low disease severity and enhancing grain yield of rice (26, 109 27

Selection of isolates for morphological and physiological characterization 120
Eighty eight isolates of Trichoderma were grouped in 49 crops with 109 locations of the 121 country. The isolates were categorized as fast, medium and slow growing on the basis of their 122 growth habit. Fifteen isolates were selected from the fast growing Trichoderma isolates on the 123 basis of different rhizosphere crops in different locations ( Table 1). 124

Spore density of different isolates of Trichoderma at 30 °C 125
Fifteen fast growing isolates of Trichoderma were observed for the number of spore per ml soil 126 at 30 °C temperature in 3 times such as 7, 14 and 21 days old culture (DOC). The number of 127 spore per ml was increased with the increasing of the age of the culture. Significant variation 128 was found among the spore density of the isolates. The highest number of spores was obtained 129 in the isolates of maize field of Nayanpur upazilla, Lalmonirhat district at 14 and 21 days old 130 culture. Another higher number of spores was recorded at 21 DOC in T 27 that was collected 131    Trichoderma isolates showed five different colony colour viz. whitish, whitish green, green, 163 dark green and light green and two types of colony consistency such as compact and loose 164 were observed at different temperatures (Table 2). 165

Influence of pH on mycelia of different isolates of Trichoderma 166
Four different pH viz., 6.0, 7.0, 8.0 and 8.5 were selected to study the influence of pH on the 167 mycelial growth on PDA among 15 isolates of Trichoderma. Fifteen isolates of Trichoderma 168 showed higher average radial mycelial growth rate at pH 6.0 and pH 7.0 among the tested 169 pH. The radial mycelial growth rate was reduced with the higher value of pH at 8.00 and 8.5.  (Table 4). 177

Molecular features of selected Trichoderma isolates 178
Four (TR 27, TR 45 ,TR 70 and TR 85 ) Trichoderma Isolates were further studied for molecular 179 characterization on the basis of their mycelial growth, morphological and physiological 180 characters at tolerable levels to high temperature and high spore density per ml. These four 181 isolates of Trichoderma were identified on the basis of their spore and mycelial structure 182

Characterization based on ITS region 208
Sequence analyses of ITS region in nucleotide PCR amplificaion at ITS4 region and ITS5 209 region resulted in 650 bp size band (Fig.04 A)

Characterization based on TEF 250
The Trichoderma isolates were also identified by sequencing of tef1 gene. PCR amplification 251 with tef1 and tef2 region showed 350 bp band (Fig. 04 B)  The phylogenetic tree differentiated the four Trichoderma isolates based on the ITS 384 sequences in two clusters (Fig. 07). Cluster I consisted of 3 isolates such as Sadar, 385 Mymensingh, Chapra, Chapai nawabganj and Nayanpur, Lalmonirhat were ascertained as T. 386 asperellum that was supported by a bootstrap value of 100% and the isolate of Sadar, 387 Moulvibazar isolate (Sub cluster II) was also noticed as T. asperellum and it was supported in 388 bootstrap of 72%. Phylogenetic tree in the Trichoderma asperellum was also divided into two 389 clusters at tef sequences whilst cluster I was grouped into two sub clusters. In cluster I, the 390 isolate of Sadar, Mymensingh was supported by 98% bootstrap value and 99% bootstrap 391 value was found in the isolate of Chapra, Chapai nawabganj. In sub-cluster II, the isolate in 392 Moulvibazar district of Bangladesh was observed, whereas the isolate of Nayanpur, 393 Lalmonirhat was grouped in cluster II with 100% bootstrap value (Fig. 08). 394

Evaluation and influence of pH on mycelia of different isolates of Trichoderma isolates 424
The highest radial mycelial growth was attained at p H 6.0 and p H 7.0 among all the isolates of 425 Trichoderma. This result was in agreement with the findings (36, 37), who reported that 426 similar results were observed with different Trichoderma isolates. The researchers (33) also 427 reported that the most favorable pH range for Trichoderma was between 5.5 and 7.5. The best 428 growth of Trichoderma virens was noted at pH 5.5 and optimal growth was between 5.0 and 429 7.0 (38). Potentiality of Trichoderma species as a bioagent that has been greatly influenced 430 by pH and most favorable pH range was 6.5-7.5 for growth and sporulation (19). 431

Preparation of soil dilution 523
Soil (1 g) was kept in a test tube with 9 ml of sterile water and stirred thoroughly for a few 524 minutes to obtain a uniform 10 ° dilute soil suspension. It was used as stock suspension. The 525 stock suspension (1 ml) was transferred with a sterile pipette into a second test tube in 9 ml 526 sterile water and it was shaken thoroughly, while a 10 -1 dilute soil suspension was obtained. 527 The same process was repeated for further dilution as 10 -3 . 528

Isolation of Trichoderma from soil and preparation of pure culture 529
Twenty ml of warm melted PDA medium was (approx. 45 °C) poured in each sterile 530 One ml of diluted soil sample (10 -3 ) was kept at the center of a plate on PDA and it was 531 spreaded with a glass rod. Each was inoculated with 1 ml of diluted sample and repeated with 532 every soil sample. The inoculated PDA plates were incubated for 7-10 days at room 533 temperature (25 ± 1 °C). Plates were observed for Trichoderma colonies after incubation of 3 534 days. The growing margin of the Trichoderma colony was cut into 5 mm discs with a cork 535 borer. The discs were carefully kept in a new PDA plates to produce a pure culture of 536 Trichoderma and the new plates were also incubated. The pure culture of Trichoderma was 537 found to grow sufficiently within 3 days of incubation. The cultures were prepared as sub-538 culture to PDA plate and were transferred to PDA slants for preservation as separate culture 539 for each. The complete grown of Trichoderma in slants was preserved in the refrigerator at 4 540 °C for further use. The isolates were identified following the key (52). 541

Morphological and physiological characterization of collected Trichoderma 542
The isolates were characterized morphologically and were purified through mycelia block 543 culture, and were maintained on acidified potato dextrose agar (APDA) medium at 28 ± 1 0 C 544 (incubation) for studying cultural characteristics (53). Radial mycelia growth of the isolates 545 was studied following the method (54). Colony diameter was measured up to 6 days after 546 inoculation of 24 hours. The colonies were filled the plant and sporulation was occurred 547 within 6 days. After 24, 48 and 72 hrs of inoculation, the radial mycelia growth was 548 measured as the mean of two perpendicular diameters and mean of three replications was 549 taken as growth of each isolate. 550 The number of colonies was recorded per gram of soil. The colonies were regular or irregular 551 in shape. Colony colors, black/ white/ grey/ blackish/ whitish/ blackish white/ whitish black 552 were recorded. After 7 days of incubation of a single culture on PDA mean radial growth of 553 the isolate was measured in the method (55). 554 Mean radial growth = Length + Breadth 2 555 Compact or loose characters were determined on the basis of compactness of the colony. 556 The surface of the culture was recorded as smooth/glistening/rough/wrinkled/ dull etc. 557 The opacity of the culture such as transparent (clear), opaque, translucent (like looking 558 through frosted glass) etc. were determined. 559 The spore density was determined using of 100 ml water in a test tube and about 10 ml was 572 poured in each PDA plate with 7, 14 and 21 days old culture of Trichoderma. The colony was 573 scraped smoothly and gently with a scalpel or slide to collect conidia. The suspension was 574 taken into a beaker from the medium and stirring was continued with a glass rod. It was 575 repeated few times. The volume of the beaker with conidial suspension was made 100 ml 576 with sterile water and 1 drop of Tween-20 was added to it and stirred to disperse well. One 577 drop of suspension of the solution was taken on the centre of haemocytometer and a cover 578 slip was kept on it. The spores/ml was counted under microscopic power of 40X with 579 haemocytometer using the block system. 580

Physiological characterization of Trichoderma 581
The isolates of Trichoderma spp. were characterized physiologically. The isolates of 582 Trichoderma were purified through mycelia block culture and were incubated on Acidified 583 Potato Dextrose Agar (APDA) medium at 30 0 C in studying cultural characteristics (53). were inoculated with the inocula of the fungus following the procedure (53). After 592 inoculation, the petri dishes were kept in the incubators for 7days. 593

Effects of pH on mycelial growth of Trichoderma 594
Four levels of p H at 6.0, 7.0, 8.0 and 8.5 were tested. PDA was prepared and three PDA plates 595 were used for each treatment. Before cooking, all ingredients were thoroughly mixed in water 596 on a magnetic stirrer. The p H of the medium was adjusted to required level with an electrode 597 p H meter. NaOH of 0.1N and 0.1N HCl were added to increase and decrease p H levels, 598 respectively. The p H level was adjusted at 6.5 after extracting of potato, while the medium 599 was boiled on a microwave oven for melting the agar powder. This media was used as 600 controlled condition. The PDA plates were inoculated with mycelial disks of the fungus. The 601 inoculated plates were incubated at 30 °C. Data on radial colony diameter was recorded. 602

Statistical analysis 603 604
The recorded data on different parameters were analyzed statistically using Web Agri Stat 605 Package computer program to find out the significant variation of experimental treatments. 606 The difference between the treatments means were evaluated with Duncan's Multiple Range 607 Test (DMRT) following the procedure (58). 608

Molecular characterization of selected Trichoderma 609
Four isolates of the 15 Trichoderma isolates were selected on the basis of their better spore 610 density and tolerable in high temperature in vitro condition. These were characterized using 611 molecular marker of ITS4, ITS5 and tef1, tef2. 612

Preparation of broth culture to harvest mycelia of Trichoderma spp. 613
Trichoderma isolates were cultured individually in 150 ml conical flasks with 100 ml liquid 614 potato dextrose culture medium for DNA extraction. Hypha were collected from potato 615 dextrose broth (PDB) with mycelia and incubated for 7 days in the incubator at 25 ± 1 0 C on 616 filter paper in a Buchner funnel, and it was washed with distilled water, frozen, and 617 lyophilized. After harvest, mycelia were wrapped with aluminum foil sheet for each isolate 618 separately and kept at 4 0 C until prior to the genomic DNA isolation. without disturbing the lower portion and 600 µl was put together in room temperature. Then 630 isopropanol was added to the supernatant and shaken slowly. Mixed by inversion was done 631 and centrifuged at 15,000 rpm for 3 minutes to produce precipitation of the cell debris. Six 632 hundred µl of 70% ethanol was mixed in blended supernatant at room temperature and 633 centrifuged at 15,000 rpm for 2 minutes. The pellet was air dried in order to evaporate the 634 ethanol. DNA rehydration solution of 25µl was added and mixed gently by finger tapping. 635 Finally the DNA solutions were preserved overnight at 4 °C. 636

Determination of DNA concentration 637
The spectrophotometer was set at260 nm for quantification of DNA. A square cuvette 638 (the zero or blank cuvette) was filled with 2 ml double distilled water and k e p t in the 639 cuvette chamber. Then the absorbance reading was adjusted to zero for 640 standardization. The test samples were prepared with 2 μl of each DNA sample in the 641 cuvette of 2 ml sterile distilled water and mixed comprehensively by pipetting. The 642 absorbance reading was taken at 260 nm and the cuvette was rinsed with sterile water. 643 The reading for each sample was recorded in the same way. The original concentration 644 was determined by using the above absorbance readings in the following formula: