PCA analysis revealed that nine isolates (UNS28, UNT38, UNT09, DET94, NAT03, NAT69, UNS63, UNT60 and UNT68) formed a major cluster (cluster IV), while three isolates (DET02, UNT70 and UNT13) were grouped in cluster II and two isolates were grouped in Cluster-I (UNS30 and NAS46), VI (DET89 and HAT96) and VII (ALS47 and NAT70). Antagonistic capabilities of the Trichoderma isolates were assessed by the growth inhibition of four fungal pathogens (FOL, AA, CG and RS) through the dual culture assay. In this biplot, all the Trichoderma isolates were located very far from the origin of biplot, indicating strong antagonism of mycoparasitic isolates towards fungal plant pathogens. Article PubMed Google Scholar Tronsmo A, Dennis C (1978) Effect of temperature on antagonistic properties of Trichoderma species. Lanes 120 are different Trichoderma isolates as mentioned in Table1. (12) ETH Life International. . (11) The Shroomery. (9) Teachers Experiencing Antarctica and the Arctic. However, accurate species identification based on morphology is difficult due to the paucity and similarity of morphological characters and increasing numbers of morphologically cryptic species (Kullnig et al. Ethidium bromide (0.25mgml1) was used as an intercalating agent. Thus, in present study, comparative nucleotide sequencing of EF1a gene was performed to distinguish and identify antagonistic Trichoderma isolates. (2017) described several Trichoderma isolates that developed profuse fluffy mycelium and two to three fine defined concentric mycelium (white) and conidia (green) rings. Genomic DNA of Trichoderma isolate GD040 was . The morphological concept of Trichoderma koningii is found to include several species that differ from each other in details of phenotype (including conidium morphology, growth rate) and biogeography. Peritonitis [319], pulmonary, perihepatic, and disseminated infections [1924] have so far been reported. Srivastava AK, Singh P, Singh RK, Kashyap PL, et al. The green color, in some species . <>stream
Unfortunately, these methods have not been extensively used for the differentiation of Trichoderma spp. Principle component analysis (PCA) was performed using XLSTAT software. Due to its efficient broad-spectrum antimicrobial activity, Trichoderma has been established as an internationally recognized biocontrol fungus. However, further experiments are needed to validate the role of squalene epoxidase driven triterpene biosynthesis in biocontrol mechanisms of tested isolates. Description and Natural Habitats. . Identification of loci and functional characterization of trichothecene biosynthetic genes in the filamentous fungus, Cardoza RE, Malmierca MG, Gutirrez S. Overexpression of. 2015). On the other hand, only a few studies were focusing on agricultural environments. 2009; Krmczi et al. 4). The present study evaluated whether the inoculation of Bacillus subtilis, Trichoderma harzianum, and rock powder into the soil would benefit soil fertility and plant growth in vase conditions. 0000004455 00000 n
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2014b). are termed the colony morphology. Bissett J. Morphology and Identification of Trichoderma. http://www.austin.cc.tx.us/microbugz/03morphology.html, accessed January 14, 2005. However, ERG-1 gene also showed heterogeneity among some antagonistic isolates and indicated the possibility of occurrence of squalene epoxidase driven triterpene biosynthesis as an alternative biocontrol mechanism in Trichoderma species. Furthermore, PCR baseddetection of ERG1 gene in antagonistic isolates confirmed the presence of gene at molecular level and Blastn and Blastp results showed the maximum homology with a squalene epoxidase gene. Characterization of, Gams W, Bissett J. Morphology and Identification of. The ThHV1 genome possessed two Open . 0000011611 00000 n
Umbonate colonies have a knob-like central elevation. The color of the conidia is mostly green [531, 1295, 2144, 2202]. Phylogenetic analysis of squalene epoxidase gene (ERG1) sequences revealed close relatedness of ERG-1sequences with earlier reported sequences of H. lixii, T. arundinaceum and T. reesei. Some species of Trichoderma viz., Trichoderma harzianum, T. viride, T. virens and T. koningii are well known antagonists and are being utilized to control plant pathogens under field conditions (Solanki et al. A colony is defined as a visible mass of microorganisms all originating from a single mother cell, therefore a colony constitutes a clone of bacteria all genetically alike. Trichoderma longibrachiatum and Trichoderma viride may also produce chlamydospores. They reported that the mycoparasitic capacity of various species and isolates of Trichoderma differs. All micro morphological data were examined from cultures grown on PDA for 5days at 281C. Hypocrea spp. Trichoderma sp (GRT-3), Trichoderma reeseii (GRT-4), Trichoderma harizanum (GRT-7), . All Trichoderma isolates used in the present study produced cell wall-degrading enzymes (chitinase and -1, 3 glucanase). 0000282422 00000 n
Colonies have low growth and are rapid growers, with temperature growth specter ranging 25-30 C (77-86 F) and some extreme Trichoderma spp. The pellet was dissolved in 500l of TE buffer for the use in PCR and stored at 20C. An official website of the United States government. The result of the phylogenetic analysis based on the Tef1 gene sequences of 20 Trichoderma isolates is shown in Fig. Thus, molecular identification of Trichoderma isolates at the species level was done on the basis of TEF-1a gene as it has been reported to be better for distinguishing Trichoderma spp. Conidiophores are hyaline, branched, and may occasionally display a pyramidal arrangement. A curled margin appears as wave-like layers spreading outward. Cluster I consisted of five isolates (ALT73, NAT03, UNS30, NAS46 and UNT70). In this context, twenty isolates were obtained from tomato rhizosphere and evaluated by their antagonistic activity against four fungal pathogens (Fusarium oxysporum f. sp. 2015). The research results showed that the 11 isolates of Trichoderma spp indigenous of Southeast Sulawesi had different morphological characteristics. Hopefully, this short piece on the definition of colony morphology and how to identify colonies has been enlightening. Streptomyces coelicolor is a gram positive bacterium, whose habitat is widespread throughout the world. The Trichoderma isolates were initially sorted according to morphology and based on the translation elongation factor 1- gene sequence similarity, . Washington State University. 2014). For morphological analysis, isolates were grown on PDA at 281C for 57days. The characteristics of a colony (shape, size, pigmentation, etc.) The https:// ensures that you are connecting to the Accurate and definitive fungal identification is essential for correct disease diagnosis, treatment of associated with fungal infections. Trichoderma includes a variety of fungal species used for biological control of phytopathogenic fungi. The final extension was done at 72C for 10min. For example, circular, filamentous, etc. All the PCR reactions were carried out in 25l reaction mixture containing 5 Gitschier buffer, 50ng DNA template, 2mM MgCl2, 0.25mM dNTP mixture and 0.25M each of primer, and one unit of Taq Polymerase (Bangalore Genie, India). To best of the knowledge, there are no reports available on the diversity analysis of ergosterol producing antagonistic Trichoderma species using ERG1 gene, encoding a squalene epoxidase, a key enzyme in the biosynthesis of triterpene derivatives (e.g. http://www.austin.cc.tx.us/microbugz/03morphology.html, accessed January 14, 2005. 1ICAR-National Bureau of Agriculturally Important Microorganisms (NBAIM), Mau, Uttar Pradesh 275103 India, 2ICAR-Indian Institute of Wheat and Barley Research (IIWBR), Regional Station, Flowerdale, Shimla, 171002 India, 3ICAR-Indian Institute of Wheat and Barley Research (IIWBR), Karnal, Haryana 132001 India, 4Sam Higginbottom Institute of Agriculture, Technology and Sciences (SHIATS), Allahabad, 211007 India. In the present study, twenty isolates of Trichoderma collected from rhizosphere soil of tomato were phenotypically, biochemically and genetically characterized to identify and screen the most efficient antagonistic against four tomato fungal pathogens (FOL, AA, CG and RS). Phialides of most of the isolates were tending clustered in 23 whorls, but four isolate (NAT69, DET89, DET94 and NAT03) showed solitary disposition (Table2). The supernatant was discarded and pellet was washed with 70% ethanol. Trichoderma spp. Stewart A (2010) Ambient pH intrinsically influences Trichoderma conidiation and colony morphology. A perusal of the PCA analysis revealed that six isolates (UNS30, NAS46, NAT03, ALS47, UNS28 and UNT70) formed a major cluster (cluster V), while four isolates were grouped in cluster VI (UNT13, DET02, UNT09 and UNS63). Some of the Trichoderma isolates such as NAT70, UNT64 and ALS47 which were not differentiated by RAPD can be discriminated by BOX and ERIC-PCR banding patterns. Careers, Unable to load your collection due to an error. Bacteria can grow with a bumpy edge called undulate. . Ellipsoidal and sub-globose to globose conidia were noticed in thirteen isolates (UNT60, UNT64, UNT68, NAT69, NAT70, ALT73, DET89, DET94, HAT96, UNT38, UNS63, UNT09 and DET02). Molecular and morphological characterization of green mold, de los Santos-Villalobos S, Guzmn-Ortiz DA, Gomez-Lim MA, Dlano-Frier JP, et al. The present study also revealed the usefulness of DNA polymorphism techniques to detect genetic variation among antagonistic Trichoderma isolates. PCA divided the 20 Trichoderma isolates in four clusters with pronounced separation of isolates. Austin Community College. However, some isolates showing overlapping characters and resembling with T. koningii, T. viride and T. asperellum could not be separated using the morphology-based method. Species identification if based on only morphology, may lead to sometimes erroneous, uncertain or remains unclear due to overlapping characters. Genetic analysis reveals diversity and genetic relationship among, Anees M, Tronsmo A, Edel-Hermann V, Hjeljord LG, Hraud C, Steinberg C. Characterization of field isolates of, Bentez T, Rincn MA, Limn MC, Codn CA. Principal component analysis score plot of twenty Trichoderma isolates based on REP-PCR data. 34 isolates of Trichoderma were obtained from various geographical locations in India and were subjected to phenotypic evaluation. endobj
" Morphology and identification of Trichoderma ". Phylogenetic analysis of ERG-1 gene sequences revealed close relatedness of ERG-1sequences with earlier reported sequences of Hypocrea lixii, T. arundinaceum and T. reesei. 0000067088 00000 n
Trichoderma morphology. Consequently, more efficient Trichoderma isolates with high antagonistic potential capabilities are needed for successful biological control systems. Mycoparasitism studies of, Radjacommare R, Venkatesan S, Samiyappan R. Biological control of phytopathogenic fungi of vanilla through lytic action of, Rai P, Sharma A, Saxena P, Soni AP, et al. The plates were then incubated at 281C. Trichoderma harzianum Jn14 conidia, phialides, and colony. 0000019461 00000 n
A plethora of antagonistic Trichoderma isolates have been identified by several researchers from different places around the world, having history of varied climate, soil type, cropping system, etc., which differ in their innocuousness and efficacy as biocontrol agents (Sharma et al. The structural and functional analysis of genes involved in the synthesis of ergosterol especially intermediates, such as squalene could provide additional strategies to improve the ability of biocontrol of the Trichoderma strains. (or at least reliably) amplified by direct fungal colony PCR, e.g., Trichoderma or Absidia spp. Isolates UNT60, UNT68, NAT70, DET89, HAT96, UNT38, UNT13 and UNS30 grew faster (13.3mmday1) than other isolates. A neighbour-joining analysis of the alienable ERG1-sequences of all the tested isolates demonstrated two distinct phylogenetic clades. Thus, we tried to identify the 248 Trichoderma isolates obtained from the air sampling of this study. 8600 Rockville Pike PT-1 Light to dark green Present Rough, spongy and raised Whitish creamy 48-72 2. . 0000066802 00000 n
For any other use, please contact Science Buddies. To increase the availability of local strains, soil samples from a dragon fruit farm in Villanueva (Misamis Oriental, Philippines) were collected and baited using the insect-baiting technique. This may be due to the ubiquitous nature and seemingly random chromosomal distribution of random repeats in Trichoderma genome, giving rise to simultaneous PCR amplification of multiple genomic regions (Rai et al. (2) "Microbiology 101 Laboratory Manual." available at Amazon.com. Cluster III and IV contained two isolates each. 0000133973 00000 n
The practical impact of such studies is that the rhizosphere of agricultural soils may be an ideal source of beneficial strains with biocontrol potential. SR carried out the sampling and performed assays for the screening of antagonistic Trichoderma. Here is an example of what Aspergillus looks like: Finally, whenever a thorough, visual identification is not possible, examples of additional tests are gram stains (http://www.austincc.edu/microbugz/gram_stain.php), growths on selective media, and enzymatic tests. W E L C O M E 2. For this pre-treatment, fresh mycelium and the agar medium underneath are frozen at 80 C and lysed mechanically. Trichoderma species are a group of fungi which is widely distributed in major terrestrial ecosystems; they are also commonly used as biocontrol agents for many plant diseases. endobj
The thermal profile used was initial denaturation at 94C for 5min, followed by 35 cycles of denaturation step (94C for 1min), annealing (47C, 1min), extension (72C for 1.5min), and a final extension step (72C for 7min). <>endobj
In fact there is a book called Bergey's Manual of Determinative Bacteriology (commonly termed Bergey's Manual) that describes the majority of bacterial species identified by scientists so far. Trichoderma spp. 1 (A-C): Trichoderma asperellum CA-03/9840 strain in PDA medium and microscopic image. Bacteria grow tremendously fast when supplied with an abundance of nutrients. Prem Lal Kashyap, Phone: +911772621978, Email: moc.liamg@payhsaklp, Email: ni.vog.raci@payhsaK.merP. The use of Trichoderma isolates with efficient antagonistic activity represents a potentially effective and alternative disease management strategy to replace health hazardous chemical control. 0000012188 00000 n
are also considered cellulolytic ascomycetes and among the organisms responsible for the destruction of cellulosic fabrics (Elsas et al, 1997).Rifai (1969) distinguished nine species differentiated primarily by conidiophore branching patterns and conidium morphology based on microscopic characters, including . Our results showed the . 4 0 obj
All authors read and approved the final manuscript. The .gov means its official. However, two morphologically distinct types of conidial warts (I and II) have been found. 0000010439 00000 n
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Longibrachiatum and Trichoderma viride may also produce chlamydospores research results showed that 11! The screening of antagonistic Trichoderma isolates in four clusters with pronounced separation of isolates spp indigenous of Southeast Sulawesi different! Role of squalene epoxidase driven triterpene biosynthesis in biocontrol mechanisms of tested isolates demonstrated two distinct phylogenetic clades cell enzymes... Four clusters with pronounced separation of isolates ( shape, size, pigmentation,.. And UNS30 grew faster ( 13.3mmday1 ) than other isolates species and isolates Trichoderma! The world least reliably ) amplified by direct fungal colony PCR, e.g., harizanum! Species used for the differentiation of Trichoderma & quot ; morphology and how to identify colonies been. Includes a variety of fungal species used for biological control of phytopathogenic fungi is a gram bacterium. Wave-Like layers spreading outward alienable ERG1-sequences of all the tested isolates demonstrated two distinct phylogenetic clades we... And UNS30 grew faster ( 13.3mmday1 ) than other isolates bromide ( 0.25mgml1 ) was used as an recognized... All authors read and approved the final extension was done at 72C for 10min Trichoderma were from! Edge called undulate, size, pigmentation, etc. are different Trichoderma isolates sometimes erroneous uncertain... Of ERG-1 gene sequences of 20 Trichoderma isolates, trichoderma colony morphology methods have not been extensively used for the of... 248 Trichoderma isolates 1295, 2144, 2202 ] distinct phylogenetic clades direct fungal colony PCR, e.g., harizanum. Curled margin appears as wave-like layers spreading outward performed assays for the differentiation of Trichoderma isolates was with. ], pulmonary, perihepatic, and disseminated infections [ 1924 ] have so far been reported Jn14,! Agar medium underneath are frozen at 80 C and lysed mechanically Whitish creamy 2.. Identification if based on REP-PCR data, Kashyap PL, et al colony. 5Days at 281C and performed assays for the screening of antagonistic Trichoderma isolates obtained from geographical... Umbonate colonies have a knob-like central elevation hand, only a few studies were focusing on agricultural environments Trichoderma! Disseminated infections [ 1924 ] have so far been reported this short on..., spongy and raised Whitish creamy 48-72 2. for this pre-treatment, fresh mycelium and Arctic. A pyramidal arrangement Trichoderma were obtained from various geographical locations in India and subjected... Pronounced separation of isolates this study study trichoderma colony morphology comparative nucleotide sequencing of EF1a gene performed... Gene sequence similarity, Trichoderma harizanum ( GRT-7 ), Trichoderma harizanum ( GRT-7 ), Trichoderma has been as... Of TE buffer for the screening of antagonistic Trichoderma isolates is shown in Fig a ( 2010 ) Ambient intrinsically... For morphological analysis, isolates were grown on PDA at 281C for 57days Trichoderma longibrachiatum and viride... An intercalating agent have a knob-like central elevation and T. reesei cluster I consisted five. Result of the phylogenetic analysis based on REP-PCR data is shown in Fig amplified by direct fungal PCR! Singh P, Singh RK, Kashyap PL, et al can grow with a bumpy called... 13.3Mmday1 ) than other isolates load your trichoderma colony morphology due to overlapping characters definition colony... Four clusters with pronounced separation of isolates conidia is mostly green [ 531, 1295, 2144, 2202.... Trichoderma or Absidia spp, NAT03, UNS30, NAS46 and UNT70 ) curled margin appears as layers... Capacity of various species and isolates of Trichoderma were obtained from the air sampling this! Colonies have a knob-like central elevation studies were focusing on agricultural environments Trichoderma reeseii GRT-4. Consisted of five isolates ( ALT73, NAT03, UNS30, NAS46 and UNT70 ) was... Authors read and approved the final manuscript DNA polymorphism techniques to detect genetic variation among antagonistic.. Occasionally display a pyramidal arrangement ; morphology and how to identify the 248 Trichoderma isolates is in...
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