Research Article - (2011) Volume 2, Issue 2
Real-time PCR based detection assay was developed for Fusarium udum, causing vascular wilt of pigeonpea. The Histone-3 gene of F. udum was targeted to design species-specific primers and probe. A comparative study was undertaken to develop a reliable and reproducible procedure for detection and quantification of F. udum from diverse samples. The sensitivity and specificity of oligonucleotides were evaluated through dot blot hybridization, standard and real-time PCR assays. The probe HFUSP showed high degree of sensitivity for DNA obtained from pure cultures of F. udum to that of environmental DNA samples. The qPCR assay specifically differentiated the F. udum from closely related species of Fusarium, other test microbes and environmental samples. The single melting curve at 84.17 and a monomorphic band of 200 bp indicates the specificity and authenticity of the PCR assays. Thus, real-time PCR assay can be used as a rapid and effective procedure that can detect minute amounts of F. udum from complex environments. Therefore, the real-time PCR assay demonstrated in the present study can be successfully used for detection and monitoring of F. udum for early infection and disease epidemiology of vascular wilt of pigeonpea.
Keywords: Fusarium udum, Histone-3 gene, SYBR green, Dot blot hybridization.
Fusarium wilt caused by Fusarium udum Butler (1906), alternate teleomorph, (Gibberella indica)  is an important biotic constraint in pigeonpea production in Indian subcontinent. In India alone, the loss due to this disease was estimated to be US $71 million per year and the percentage of disease incidence varies from 5.3 to 22.6% . Identification and detection of pathogenic Fusarium sp. was traditionally based on either symptom on the host or culture dependent isolation of the pathogen from affected host tissue . These classical approaches are becoming increasingly problematic because more than one forma specialis may occur on a given host, along with non-pathogenic, common soil and rhizosphere inhabitants . In addition, the morphological identification is not feasible to quantify pathogen load in different plant tissues during the growing season or in commodities after harvest. Moreover, isolation and culture dependent enumeration may introduce a bias in favor of faster-growing species .
PCR-based methods have been reported for the detection of soilborne Fusarium species [7,9,10,23,30]. However, with advent of realtime PCR (=RT-PCR), plant pathologists possess the unprecedented ability to accurately quantify a specific pathogen within a host plant . Since last one decade efforts have been made in detection and quantification of various plant pathogens [29,24,2]. For instance, RTPCR with SYBR green chemistry for quantification of F. solani f.sp. phaseoli in both sterile and non-sterile soil  and RT-PCR based on TaqMan probe chemistry to quantify the F. culmorum in infected plant tissue .
Nuclear rRNA, including the small and large subunits and the internal transcribed spacer (ITS) region, proved as an ideal target for the detection of different isolates/species of Fusarium such as F. oxysporum f. sp. vasinfectum, F. oxysporum f.sp. ciceri and F. sporotrichioides [26,16,20,12]. Besides ITS region, several researchers applied other conserved genes such as translational elongation factor 1- α (TEF1-α) [13,4,28] Cellobiohydrolase-C , Histone-3  and Topoisomerase-II  for development of species-specific PCR primers. In recent studies, primer set were developed targeting to esyn- 1 gene encoding multifunctional enzyme enniatin synthetase to identify enniatin-producing Fusarium species . Similarly, toxin producing genes were also targeted to design species specific ologonucleotides for identification, detection and quantification of different pathogenic Fusarium species [33,19].
Though Yadav et al.  recently developed Topoisomerase-II gene based real-time PCR for identification and detection of F. udum, those assays were not validate with F. acutatum, another important wilt causing pigeonpea pathogen, which is almost undistinguishable morphologically with F. udum . Keeping in view the present study was aimed to develop a rapid and sensitive RT-PCR assay using species-specific oligonucleotides complementary to the H3 gene for detecting F. udum and to evaluate these assays on diverse samples such as, artificially infected seedlings, plant materials and agricultural field samples. The specificity and suitability of these species-specific oligonucleotides were validated by using dot-blot hybridization, conventional and real-time PCR.
Fungal isolates and culture conditions
Different pathogenic isolates of F. udum including four reference cultures from different geographical origin infective on three different alternate hosts viz, Cicer arietinum (241426), Crotalaria verrucosa (170431), Cajanus indicus (193652) and Cajanus cajan (1708) were obtained from the Centre for Agriculture and Biosciences International (CABI), U.K and National Agriculturally Important Microorganism Culture Collection (NAIMCC), India (Table 1). These cultures were referred all throughout the paper for validation. In all validation experiments, different tester microbes obtained from, India were used (Table 2). All fungal cultures were grown on potato dextrose agar (PDA) plates at 25±2°C while bacterial cultures were maintained on nutrient agar (NA) plates.
|Sl. No.||Accession No.||Biological origin||Geographical location|
||CABI 241426||Cicer arietinum||Hyderabad, Andhra Pradesh|
||CABI 170431||Crotalaria verrucosa||Hyderabad, Andhra Pradesh|
||CABI 193652||Cajanus indicus||Hyderabad, Andhra Pradesh|
||NAIMCC-F-1708||Cajanus cajan (Stem)||Kanpur, Uttar Pradesh|
||NAIMCC-F-1703||Cajanus cajan (Stem)||Fatehpur, Uttar Pradesh|
||NAIMCC-F-556||Cajanus cajan (Stem)||Dholpur, Rajasthan|
||NAIMCC-F-557||Cajanus cajan (Stem)||Baran, Rajasthan|
||NAIMCC-F-563||Cajanus cajan (Stem)||Rohtak, Haryana|
||NAIMCC-F-1688||Cajanus cajan (Stem)||Banda, Uttar Pradesh|
||NAIMCC-F-234||Cajanus cajan (Stem)||Andhra Pradesh|
||NAIMCC-F-1686||Cajanus cajan (Stem)||Banda, Uttar Pradesh|
||NAIMCC-F-567||Cajanus cajan (Stem)||Bhiwani, Central Haryana|
||NAIMCC-F-1713||Cajanus cajan (Stem)||Varansi, Uttar Pradesh|
||NAIMCC-F-1687||Cajanus cajan (Stem)||Banda, Uttar Pradesh|
||NAIMCC-F-564||Cajanus cajan (Stem)||Thazzar, Eastern Haryana|
||NAIMCC-F-1211||Cajanus cajan (Root)||Tandawa, Jharkhand|
||NAIMCC-F-559||Cajanus cajan (Stem)||Faridkat, Central Punjab|
||NAIMCC-F-1213||Cajanus cajan (Stem)||Sangbaria, Jharkhand|
||NAIMCC-F-1215||Cajanus cajan (Stem)||Maurshidabad, West Bengal|
||NAIMCC-F-571||Cajanus cajan (Stem)||Faridabad, Haryana|
Table 1: Cultures of F. udum collected from different hosts and various geographical locations of India.
|Sl. No.||Name of the microorganism||NBAIM accession no.|
|1||Fusarium acutatum||NAIMCC-F-00760, CABI 375327|
|3||Fusarium oxysporum ciceri||NAIMCC-F-00858, CABI 241424|
|4||Fusarium oxysporum ciceri||NAIMCC-F-00857, CABI 259237|
|5||Fusarium oxysporum f.sp. carthami||NAIMCC-F-00833|
|6||Fusarium oxysporum f.sp. carthami||NAIMCC-F-00834|
Table 2: Different groups of microorganisms used for the testing of specific oligonucleotide primers and probe.
Preparation of seed samples
Conidial suspensions were prepared by culturing F. udum (193652) on PDA plates at 25±2°C under dark for 10 days. The surface of the fully grown culture plate was flooded with 2 mL of sterile distilled water (SDW) and agitated for 2 min. The conidial suspension was serially diluted to adjust 5×106 conidia mL-1 using haemocytometer. Pigeonpea seeds were disinfected with 1% NaOCl, followed by rinsing twice with SDW and seeds were soaked for 1h in a conidial suspension at different dilutions. To enrich the fungal culture on seed surface, the healthy seeds were placed in a sterile test tube (10 seeds tube per tube), mixed with 0.5 mL of liquid culture of MDP medium (2% malt extract, 2% dextrose, 0.1% peptone) and incubated at 25°C for 48h. The tubes were vortexed briefly and seed free suspension (mycelia and conidia) was collected to microcentrifuge tube through millipore filtration (0.45 μm).
Pigeonpea seeds (susceptible cultivar T-20) were sown in different pots (15 cm diameter) filled with garden soil. The pots were watered daily and maintained at 22±2°C with a 14h photoperiod in a greenhouse. Three weeks after sowing, the plants were injected with 50 μL of conidial suspension (5 × 106 conidia mL-1) at the base of the stem. After 40 days of inoculation, root and stem samples from the plants showing typical symptoms of the infection were harvested. Three replicates were maintained for each set of experiment.
Extraction of Genomic DNA
Total genomic DNA, from fungal cultures was isolated using the protocol described earlier Babu et al. (2007). DNA from bacteria and actinomycetes was extracted using Wizard Genomic DNA Purification Kit (Promega, USA). For isolation of genomic DNA from F. udum infested soils and pigeonpea plant materials (artificially and naturally collected samples) was carried by using MoBio soil DNA isolation kit (MoBio Inc., CA) and DNeasy plant DNA extraction kit (Qiagen Inc., Canada) respectively and the samples were stored at -20°C for further use.
PCR amplification and sequencing of Histone-3 gene
The Histone-3 (H3) gene was amplified using the primer pair H3-1a (5’-ACTAAGCAGACCGCCCGCAGG-3’) and H3-1b (5’-GCGGGCGAGCTGGATGTCCTT-3’) and PCR was carried out in Dyad Peltier thermal cycler (BioRad, USA) in a 25μL volume with conditions as described earlier . PCR amplified products were resolved on a 1.2% agarose gel containing ethidium bromide (0.2 μg mL-1) and visualized under UV light. The amplified products from four reference strains of F. udum were gel extracted, purified and subjected for direct sequencing on an ABI automated DNA Sequencer using ABI Big Dye termination cycle sequencing ready reaction kit, following the manufacturers protocol.
Sequence analysis and design of specific oligonucleotides
All the H3 gene sequences were compared with the available H3 gene sequences in the GenBank database using BLASTn program. Multiple H3 gene sequences from F. udum (241426, 170431, 193652, 1708) and other H3 gene reference sequences retrieved from GenBank database were aligned using Gene Doc (version 2.6.002) . The sequence alignment was visually checked for regions having similarity among the isolates of F. udum and variable in those of other species. The specific locations thus obtained were subjected for Primer3 online software  to design a pair of PCR primers and a single oligonucleotide probe. The forward (HFUSF) and reverse (HFUSR1) primers were evaluated in silico to yield a product of 200 bp size. The theoretical specificity of the three oligonucleotides was checked against sequences of closely related fungi in GenBank. Parameters such as the % G+C content and the absence of self complementarity in oligonucleotides and complementarity between primers were analyzed using the computer program Gene Runner (Hastings Software, USA). The oligonucleotides were custom synthesized by Bangalore Genei, India.
Validation of the primers in standard PCR
The designed species-specific primers HFUSF and HFUSR1 were used for PCR amplification of F. udum isolates, other representative test microbes, infected seedlings, plant materials and agricultural field samples (Table 1-3). PCR protocol was standardized with variable cycle numbers, annealing temperatures and different concentrations of target DNA. The amplifications were carried out (25μL) by mixing 2.5μL of 10X PCR buffer, 0.2mM, 2.5mM each dNTPs, 2μL of 5% glycerol, 0.5μL of 10 mg mL-1 BSA, 0.5μL of 5% DMSO, 5pmol of each primer, 1 U of Taq DNA polymerase and 35ng of template DNA. The PCR reaction performed for 40 cycles of denaturation at 95°C for 1 min, followed by annealing at 54°C for 10 s, extension at 72°C for 20s and the final extension step of 72°C for 10 min.
Dot blot hybridization assay
The oligonucleotide probe HFUSP (3’-CGCCACATCAACCACAGCTCAACACT-5’) was labelled with digoxigenin-3-O-methylcarbonyl-amino-caproyl-5-(3-aminoallyl)- uridine-5-triphosphate (DIG-11-dUTP) using DIG DNA labeling kit (Roche Diagnostics, Germany), following manufacturer’s instructions. Initially, the DIG labelled probe specificity was tested with the genomic DNA of different strains of F. udum (Table 1). Later the dot blot hybridization assay was carried out for genomic DNA from different test microbes and agricultural field samples (Tables 2 & 3) as unknown target and genomic DNA from four reference strains of F. udum were used as control. All target DNA samples irrespective of their concentrations (4-8μL) were blotted manually onto positively charged nylon membrane (Boehringer Mannheim). Pre-hybridization treatments for membrane such as denaturation, neutralization and cross-linking were carried out as described by . The assay was optimized by adjusting the conditions like: hybridization temperature, concentration of the probe (0.5–2 pmol mL-1) and salt concentration of the wash buffer were determined as described elsewhere . Detection of the hybridized DIG labeled probes were carried out and then visualized with the colorimetric substrates NBT/BCIP. Dot blot hybridization assay was performed at least thrice under the optimized conditions.
|Name of the DNA sample||Dot blot hybridization||Conventional
|Mean of CT value||Log of DNA
|Pure culture #||+||+||14.00||100|
|Assay for Artificially infected samples|
|Plant root (IPR1-3)||−||+||21.75||1.5|
|Plant stem (IPS1-3)||weak signal||+||19.25||6.5|
|Assay for Agricultural field samples|
|Plant root (APR1-3)||−||+||27.50||0.002|
|Plant stem (APS1-3)||−||+||26.75||0.021|
|Rhizosphere soil (ARS1-3)||−||+||33.25||0.0015|
*-5×106 conidia mL-1, + presence, − absence, #- F. udum 193652
Table 3: Evaluation of species specific probe and primers under three different assays.
Specific real-time PCR assay for F. udum
RT-PCR assay was performed with Step-One™ real-time PCR system (Applied Biosystems, USA) using SYBR green fluorescent molecules. All reactions were performed in 0.5mL thin-walled, optical grade PCR tubes with a reaction volume of 20μL. The reaction mixture consists of 10μL of 2X SYBR® Green I Master Mix, 900nM of each primer (HFUSF and HFUSR1), 2μL of DNA template and water to the final volume. The amplification conditions of the reaction were set at 95°C (10 min), 40 cycles at 94°C (15s), 54°C (10 s), and 72°C (20s), and fluorescence read at 72°C at the end of each cycle, followed by final melting curve analysis at 65-95°C with an increment of 0.1°C s-1. Determination of cycle threshold (CT) and data analysis were carried out with the help of Sequence Detection System Software v.1.2, provided by the Applied Biosystems, USA. The specificity of SYBR green assay was verified using different F. udum isolates obtained from various agro-climatic regions, along with other tester microbes and agricultural field samples (Tables 1-3). The DNA extractions were preformed as described above. Template DNA (5-60ng) added to the reaction mixture, F. udum genomic DNA used as positive control and DNA from other test microbes and agricultural field samples were used as unknown targets.
Sensitivity and standard curve analysis
Genomic DNA from F. udum (241426) with an estimated initial concentration of 100ng μL-1 was serially diluted (1:10) with SDW. The results were analyzed by plotting the Log of template concentration against CT values. The sensitivity or minimum detection limit of the assay was estimated so as to quantify and detect the lowest amount of target DNA, when the cycle threshold was being attained up to 40 cycles.
Detection of F. udum in seedlings, plant and agricultural samples
Artificially infected seed and plant samples were prepared and their DNA was extracted as mentioned above. Fusarium infected pigeonpea plant and rhizospheric soil samples were collected from long-term pigeonpea growing agricultural fields of Kusmaur (25°53’57”N, 83°29’01”E), Mau district, Uttar Pradesh, India. The infected rhizosphere soil and plant material DNA was extracted as mentioned above. The extracted DNA from each sample was used as unknown targets for identification and detection of F. udum. Genomic DNA of F. udum (193652) was used as positive control while healthy and uninfected (pigeonpea) plant DNA was used as negative control. The RT-PCR assay for standard graph was performed under the similar conditions as described above.
Histone-3 amplification and sequence analysis
Partial Histone-3 gene (nearly about 550 bp) was amplified from all the isolates of F. udum using H3 universal fungal primers. The partial sequence of H3 gene consists of three exons, which were separated by two intron regions (Figure 1). Multiple sequence alignment of H3 region of the four representative isolates with other related fungal species revealed that the two intron regions were conserved in F. udum isolates and variable among the other related fungi. The edited H3 gene sequences have been deposited in GenBank and accession numbers were obtained (GQ289554 – GQ289557). After editing and realignment of H3 gene sequence the two intron regions were selected for development of species-specific oligonucleotides. The forward primer HFUSF (5’-ATCATCACTAACTTCATCACCAAT-3’) was designed from intron-I region, while reverse primer HFUSR1 (3’-TGTCGAATGTTAGTAAGTGTTG-5’) and one probe of 26mer HFUSP (3’-CGCCACATCAACCACAGCTCAACACT-5’) were designed from intron-II (Figure 2).
Figure 2: Development of specific oligonucleotide primers and probe: Alignment of partial H3 sequences from four isolates of F. udum (in this study) and other Fusarium reference sequences were taken from GenBank. Nucleotides are shown in colour bars (A-red, G-yellow, T-blue and C-green). The solid line rectangles indicate specific nucleotide regions used for the development of specific oligonucleotide primers and the dashed rectangles shows the specific region used for designing of the probe.
Specificity of primers by standard PCR
In conventional PCR all the isolates of F. udum, artificially infected seedlings, infested plant and soil samples (artificially and naturally) from green house and agricultural field samples of pigeonpea yielded a single amplified product of 200bp (Figure 3). The primers were specific for F. udum as none of the other tester microbes exhibited any amplification (Figure 4, Table 2).
Figure 3: Species specific PCR Amplification of H3 gene cluster: Primers HFUSF and HFUSR1 were used for amplification of 200 bp fragment of H3 gene cluster of F. udum from different samples. (a) Lane 1 to 20 are F. udum isolates listed in the Table 1, (b) Lane 21-conidial suspension, Lane 22-pure culture, Lane 23,24 infected seedlings (ISD1-3), Lane 26–28 infected plant root (IPR1- 3), Lane 29–31 Infected plant stem (IPS1-3), Lane 32–34 Agricultural plant root (APR1-3), Lane 35–37 Agricultural plant stem (APS1-3), Lane 37–40 Agricultural rhizosphere soils (ARS1-3) as listed in Table 3. M-1 kb molecular ladder.
Conditions for specific hybridization of probe
Detectable hybridization signals were obtained with the oligonucleotide probe at 1.5pmol mL-1 concentration; and optimum hybridization temperature was 43°C for 15h. The variable parameters were fixed at optimum as mention: membrane washing twice, 5 min per wash, in 2X SSC containing 0.1% of SDS, then twice, 5 min per wash, in 0.1× SSC, 0.1% SDS, followed by 15 min per wash per two times, in 0.1× SSC containing 0.1% SDS (20±2°C). The dot blot assay carried out with 5μL of genomic DNA allowed the specific detection of all the F. udum strains. Probe HFUSP selectively hybridized with strains of F. udum but failed to do so with all other tester microbes (Figure 5; Table 2). In case of DNA samples obtained from artificially infected seedlings, plant material and naturally infected field samples the probe fails to produce detectable signals, exception with plant sample (IPS1) where a weak signal was obtained (Data not shown) (Table 3).
Sensitivity and standard curve analysis
Under optimized conditions the RT-PCR assay showed standard fluorescence amplification representing exponential growth of PCR products and a standard curve was obtained at least 6 orders of magnitude. The lowest detection limit of the assay was 1 pg, obtained at CT value of 34.0. The standard curve revealed that the primer set used in the present study was quite accurate over a linear range and high correlations between CT and DNA quantities were obtained with a slope of -3.387 and R2 =1. Amplification efficiency of the target gene was 97% among all the samples used in this study (Figure 6a). The mean CT values recorded for the DNA samples obtained from reference strain of F. udum, conidial suspension, artificially infected pigeonpea seedlings, plant materials (root and stem), and naturally obtained diseased pigeonpea plants and rhizospheric soil batches ranged from 14.00 to 33.25. While the mean limit of detection ranged from1.5 pg to 100ng (Table 3). The real time PCR SYBR green assay enabled specific detection of F. udum with a single melting curve at 84.17°C without cross detecting any of the representative tester microbes (Figure 6b).
Figure 6: Standard curve Analysis: a. Standard curve of F. udum (CABI 241426) DNA concentration standards against the cycle threshold (CT). b. Melting Curve Analysis: Melting temperature (Tm) at 84.17 with a single peak indicates the specificity of the species specific the primers HFUSK and HFUSR1 to F. udum.
Molecular diagnostics for plant pathogens are important to detect fungal contaminants in crops, commodities and also act as valuable tools for plant quarantine purposes [5,38]. However, quantitative estimation of a pathogenic population is also essential to investigate the ecology and epidemiology of disease, which leads pathologists to monitor harmful microbial populations spread and distribution over time and space . In particular, when plant diseases caused by any member of Fusaria species complex, it is very crucial to diagnose or quantify such an individual population to develop disease management strategies and breeding for resistance programs. Moreover, accurate and rapid tools are required to investigate and monitor the global spreading and disease aggressiveness. Early identification of pathogen is very crucial for framing disease management strategies and breeding programs. For example, selection and breeding for resistance in cereals are facilitated by the use of species specific markers to diagnose the various fungi implicated in Fusarium ear blight [30,34]. The situation for F. udum is equally complex as this species was associated with a wide range of diseases in diverse plant species under diverse agroclimatic regions .
In the present study, we targeted on rRNA, β-tubulin, TEF1-α and Histone-3 genes to design and develop species-specific probe and primers for F. udum. Sequencing of each gene, followed by multiple sequence alignment revealed that the rRNA and β-tubulin genes exhibited minimum variability to differentiate Fusarium sp. to that of F. udum. Further, the sequence alignment of TEF1-α have multiple variations within the isolates of F.udum (data not shown). Therefore, these genes were not considered as suitable targets to design speciesspecific primers and probes. However, partial sequences of Histone-3 gene showed that this gene was conserved among the isolates of F. udum and showed sufficient variability amongst the Fusarium sp. The Histone-3 gene was conserved among the isolates of F. udum and showed significant variability with F. oxysporum sp. ciceri. Moreover, in BLASTn analysis of the primers and probe showed 100% similarity with G. indica, an alternate teleomorph of F. udum  while no significant similarity was observed with its closely related F. xylarioides and F. acutatum sequences . Thus, oligonucleotide primers (HFUSF and HFUSR1) and probe (HFUSP) can be used for specific amplification and detection of F. udum and its telomorph G.indica.
The genetic diversity analysis of F. udum revealed high degree of genetic diversity among populations . Therefore, in present study, the real-time PCR assays have been validated with many target organisms along with tester microbes. Similarly, in our previous study also we have been included an array of target species to validated specificity of the real-time PCR assays . PCR Optimization especially of the annealing temperature at 54°C for 10s allowed specific amplification of F. udum among all the isolates analyzed. Thus, the protocol developed in our study was tested with twenty different isolates of F. udum from various biological and geographical origins. Further, in dot bolt assay the probe HFUSP showed high specificity towards the target DNA and produced strong signals with pure cultures expect with agricultural field sample (IPS1), where weak signals were obtained. This might be because of the low concentrations or may be due to the short length of the probe (26 mer). On the other hand the probe did not show any false positive or non-specific hybridization with other representative microbial isolates (Table 2). Our results clearly demonstrated that F. udum could be differentiated from that of other Fusarium species, using these specific primers and probe. Recently, several researchers demonstrated this approach for the detection of Fusarium sp. in different hosts [25,28]. As evident from the RT-PCR studies, the sample IPS1 consists of 6.5ng μL-1of targeted DNA (Table 3), while a weak signal was observed on dot blot analysis. The efficiency may be used as a rough estimate for how well real-time PCR works in a given sample type. When the results of the H3-targeted dot-blot hybridization was compared, a reasonable level of sensitivity was obtained using a standard conventional PCR, while real-time PCR had a superior sensitivity and also proved to be a more convenient and less expensive method for identification and quantification of F. udum populations. Thus, the developed RTPCR assay can be considered as far more superior and novel tool for detecting F. udum especially from that of its closely related species like F. acutatum infecting the same host and also for quantification of this pathogen in soil and plant tissue at picogram level of target DNA. The specific primers and probe generated in the present study could be further exploited in epidemiological studies of F. udum. In conclusion, the novel detection system developed in the present study will be a useful tool in monitoring early infection of F. udum in disease outbreaks and can be helpful for formers to administrate disease management practices.
We thank Indian Council of Agricultural Research, New Delhi, India for providing financial assistance under the Application of Microbes in Agriculture and Allied Sectors (AMAAS) project.