Molecular characterization of Chaetomium species using URP-PCR

Chaetomium spp. are common colonizers of soil and cellulose-containing substrates. Seventeen isolates of Chaetomium spp., which included 15 isolates of C. globosum and one each of C. reflexum and C. perlucidum, were genetically characterized with universal rice primers (URP primers derived from DNA repeat sequences in the rice genome) using polymerase chain reaction (URP-PCR). Out of the 12 URP’s used in the study, nine primers were effective in producing polymorphic fingerprint patterns from DNA of Chaetomium spp. Analysis of the entire fingerprint profile using the unweighted pair-group method with arithmetic averages (UPGMA) clearly differentiated C. globosum isolates from C. perlucidum and C. reflexum. One of the primers, URP-2R, produced a uniform DNA band of 1.9 kb in all the isolates of C. globosum but not in C. perlucidum and C. reflexum, which can be used as molecular marker to differentiate C. globosum from other species. Our results indicate that URP’s are sensitive and give reproducible results for assaying the genetic variability in Chaetomium spp.

Chaetomium is a genus belonging to the class Pyrenomycetes (Ascomycotina), order Sordariales and family Chaetomiaceae.It is found extensively on various agricultural commodities.The genus can produce an Acremonium-like state (imperfect stage) on culture media and is characterized by superficial flask-shaped perithecia, which are surrounded by dark, stiff hairs.C. globosum is one of the commonest species growing saprophytically in the rhizosphere and phyllosphere.It is a common colonizer of soil and cellulose-containing substrates and has been reported to be a potential biocontrol agent.C. globosum has been reported effective in minimizing damage caused by seed rot and damping off, due to several seed-borne and soil-borne plant pathogens like Pythium ultimum, Alternaria raphani, A. brassicol and Fusarium spp.(Harman et al., 1978;Vannacci and Harman, 1987).
The seedling blight caused by Rhizoctonia solani has been successfully controlled by seed treatment with Chaetomium spp.(Baker, 1968).C. globosum has also shown an antagonistic effect against rice blast (Pyricularia oryzae) (Soyton and Quimio, 1989).Our recent studies have indicated its bioefficacy in controlling spot blotch of wheat caused by Cochliobolus sativus (Aggarwal et al., 2004).Biochemical characterization of the fungus has shown the production of β 1,3-glucanase and xylanase (Ahammed SK, Ph.D. Thesis, Indian Agricultural Research Institute, New Delhi, India, 2003;Ahammed et al., 2008).C. globosum has a great potential as a biocontrol agent and has been classified based on morphological descriptions of colony growth and perithecia (Millner et al., 1997;Ahammed et al., 2004;2005a), but this is not sufficient and a significant variation needs to be defined for each strain at the molecular level.C. reflexum and C. perlucidum are two other species, morphologically very similar to C. globosum.Therefore, there is a need for molecular markers to characterize differences at the inter and intraspecific level.
Repeat sequences from Korean weedy rice, originally referred to as universal rice primer (URP), have been used for the fingerprinting of diverse genomes of plants, animals and microbes (Kang et al., 2002), but only very few fungi (Kang et al., 2001;Kang et al., 2002;Jana et al., 2005).Chaetomium globosum isolates had been characterized earlier by the PCR-RAPD technique (Ahammed et al., 2005b).However, the use of repetitive sequences derived from plant genomes as molecular markers has not received attention in the fingerprinting of this fungus.The objective of our study was to use URP's for generating DNA fingerprint profiles of different C. globosum isolates and compare them with C. reflexum and C. perlucidum, in order to genetically differentiate the species.
Pure cultures of 15 Chaetomium globosum isolates (Cg1-Cg15) and one isolate each of C. reflexum (Cr) and C. perlucidum (Cp) collected from various locations of India were established.For DNA isolation, the cultures were grown in potato dextrose broth (PDB; pH 5.5) for 7 days at 28 ± 1 °C in a shaker incubator.Mycelia were filtered through filter paper (Whatman n. 1) and DNA was extracted using the cetyltrimethyl ammonium bromide (CTAB) method (Murray and Thompson, 1980).The mycelium was ground in liquid nitrogen, transferred to DNA extraction buffer (0.1 M Tris, 1.5 M NaCl, 0.01 M EDTA) and kept at 65 °C for one hour with occasional stirring.Equal volumes of chloroform:isoamyl alcohol (24:1) were added to all tubes, followed by centrifugation.The upper aqueous phase obtained by precipitation with 0.6 th volume of ice-cold isopropanol was again centrifuged.The pellet was washed with 70% ethanol and dried at room temperature.Finally, the nucleic acid was dissolved in TE and stored at -20 °C.
URP's are primers with 20 oligonucleotides each, originally obtained from repeat elements of weedy rice by Kang et al. (2002).There are 12 URP primers which were synthesized by Genuine Chemical Corporation (GCC), India.PCR was performed in a Temperature Gradient Thermal Cycler (BioRAD, USA).Concentrations of DNA template, primer and deoxynucleotide triphosphates (dNTPs) and the optimum annealing temperature were standardized for each primer in preliminary trials to obtain DNA fingerprint profiles (Table 1).Each PCR reaction contained 50-100 ng of genomic DNA, 200 μM of each dNTP (dATP, dGTP, dCTP and dTTP), 0.2 μM primer, 1.5 mM MgCl 2 , 2.5 U Taq DNA polymerase, and 1X Taq buffer in a total reaction volume of 25 μL.Thermal cycling conditions were: initial denaturation at 94 °C for 4 min, followed by 35 denaturation cycles at 94 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 2 min.A final extension step at 72 °C for 7 min was also performed.
The URP -PCR products were electrophoresed on a 1.2% agarose gel in TBE buffer, visualized by staining with ethidium bromide and photographed using a Gene Genius Gel Documentation System (Syngene Inc, Cambridge, UK).
Relatedness among the 17 isolates of Chaetomium spp. was estimated by means of scorable DNA bands amplified from different URP markers.Each band was considered as character and was scored as either present (coded as 1) or absent (coded as 0).Cluster analysis with the unweighted pair group method with an arithmetic average (UPGMA) algorithm was performed using NTSYS-PC (v.2.01; Rohlf, 1998) to produce a dendrogram.The experiments were repeated three times, and each time identical results were obtained.
Twelve oligonucleotide primers (URP's) were used for the molecular analysis of 15 isolates of Chaetomium globosum and one isolate each of C. reflexum and C. perlucidum.These isolates were used as templates to assess the wide distribution of the URP nucleotide motif sequences in the genome of Chaetomium spp.Nine URP's out of 12 gave good amplification in all the C. globosum, C. reflexum and C. perlucidum isolates used in this study.Different levels of polymorphism were obtained with these primers (Table 1).Amplification products for all primers were polymorphic, except for URP-30F, which produced a monomorphic band.The amplified DNA bands ranged from 250 bp to 3000 bp for each isolate.
Amplification with primer URP-2R produced a uniform DNA band of 1.9 kb in all the isolates of C. globosum, but not in C. reflexum and C. perlucidum (Figure 1a).The data on banding pattern with this primer was analyzed, and the dendrogram obtained showed the formation of two major clusters with C. perlucidum separating out singly, showing only 33% similarity with the C. globosum and C. reflexum isolates.In cluster I, there was 100% similarity 944 Aggarwal et al.Phylogenetic analysis of a combined data set obtained from nine URP's showed formation of two main clusters with only 65.3% similarity between them (Figure 2).The dendrogram showed high genetic similarity among different isolates of C. globosum obtained from different sources.Cluster I consisted of isolates Cg1, Cg9, Cg4, Cg5, Cg6, Cg7 and C. perlucidum and C. reflexum.Bootstrap analysis indicated that, within this cluster, isolates Cg4 and Cg6 showed 53.9% similarity, and these two isolates showed 92.4% similarity with Cg5.Isolate Cg9 and C. reflexum formed a separate group within cluster I, separating out at a bootstrap value of 21 from the other isolates and C. perlucidum.Within this cluster, isolates Cg4 and Cg5 showed 93% similarity.The other C. globosum isolates were grouped into cluster II, in which Cg2 and Cg3 showed a high bootstrap value (98.2%), forming a separate subgroup within this cluster.This group showed > 63% genetic similarity among the isolates.Isolate Cg8 separated out from this other subgroup within this cluster at the bootstrap value of 54.6%, having the rest of the isolates viz., Cg10, Cg11, Cg12, Cg13, Cg14 and Cg15.In our earlier studies on molecular characterization of C. globosum using RAPD primers, nine isolates were grouped into two distinct clusters, with isolates Cg2, Cg3 and Cg4 in one cluster and the remaining isolates in another (Ahammed et al., 2005b).Genetic diversity using AFLP markers in this fungus was also explored earlier (Aggarwal et al., 2003), showing that five C. globosum isolates formed two distinct clusters, one comprising isolates Cg6, Cg7 and Cg8, and the other encompassing isolates Cg1 and Cg5.The use of URP markers in the present study enabled us to distinguish 15 different C. globosum isolates collected from different sources, and also from C. reflexum and C. perlucidum.Previous studies have indicated that these rice repeat sequences based on which the URP primers were designed are conserved in fungi too, enabling us to detect the variations at the interspecific and intraspecific levels.There is one previous report on the use of URP's for the genetic differentiation of a fungus, Macrophomina phaseolina (Jana et al., 2005).Our analysis revealed that 75% of the URP's could amplify the scorable and reproducible bands with genomic DNA of Chaetomium globosum, C. perlucidum and C. reflexum isolates.Primers URP-6R, URP-4R and URP-2R produced distinct amplification profiles among the Chaetomium spp.isolates used in the present study and were therefore considered the best markers.All URP primers, except URP-30F, revealed more polymorphism in our study, although  the results of Kang et al. (2001Kang et al. ( , 2002) ) and Jana et al. (2005) showed more polymorphism with primers having a GC content > 50%.Primers URP-13 R, URP-32F and URP-38F (Kang et al., 2002) did not amplify the genomic DNA of Chaetomium spp. in this study.The genetic differentiation of C. globosum isolates has a great significance, considering that our earlier findings have indicated the potentiality of this fungus as a biocontrol agent (Aggarwal et al., 2004).The present investigation shows that URP-PCR fingerprinting is a valuable tool for rapid identification and differentiation of fungal strains.PCR fingerprints have shown high DNA polymorphism between strains of the same species and even between different Chaetomium spp.Moreover, our purpose of using URP markers was to differentiate the strains of C. globosum and also to develop species-specific markers.Amplification with URP-2R produced a uniform band of 1.9 kb in all the C. globosum isolates, but not in C. perlucidum and C reflexum.Further work on cloning and sequencing of this amplicon to develop a SCAR marker is in progress.
between isolates Cg2 and Cg 8, Cg12 and Cg 13, and Cg 10 and Cg 11, while in cluster II, Cg4, Cg5, Cg6 and Cg7 exhibited 100% similarity.The C. reflexum isolate separated out in this cluster, showing 82% similarity with the above isolates and with the C. globosum isolate Cg3 (Figure1b).A DNA band of 1300 bp was specifically amplified by primer URP-6R only in C. reflexum and not in any of the C. globosum and C. perlucidum isolates (Results not shown).On the other hand, URP-4R amplified a band of 1200 bp specific to C. perlucidum and not present in any isolate of C. globosum and C. reflexum (Results not shown).The analysis based on individual URP primers (URP-2R, URP-4R and URP-6R) indicated a high level of genetic similarity between isolates Cg4, Cg6, Cg7 and Cg9 in one cluster and Cg10, Cg11, Cg12, Cg13, Cg14 and Cg15 in a separate cluster, in spite of the fact that they were obtained from different geographical regions of India.

Figure 2 -
Figure 2 -Dendrogram obtained after combined bootstrap analysis.The numbers at the forks show the percentage of times the group consisting of the species which are to the right of that fork occurred.Cg = Chaetomium globosum; Cr = C. reflexum; Cp = C. perlucidum.

Table 1 -
Sequences of Universal Rice Primers (URP's) and polymorphism obtained in Chaetomium spp.