Introduction

The cereal crop rice (Oryza sativa L.) is the staple food of over half of the world’s population1,2 that provides more than 50% of caloric intake3. Rice kernel smut (RKS) is caused by fungal pathogen Tilletia horrida that was first described in Japan in 1896. It causes a partial bunt that affects both yield and quality of rice by producing black, sooty masses of powdery spores which replace all or part of the grain. The pathogen primarily exists in nature as dark-brown tuberculate teliospores that are widespread in soil overwinter and outside or inside of the host plant seeds. The spores can survive for more than 1 year in soil and for 3 year inside the seed4,5,6. Prior symptom scouting for RKS is not feasible. When symptoms of this disease are identified as present in a farmer’s field, it is too late to apply fungicide for control7,8.

Previously, the rice kernel disease was considered as a persistent but minor disease5,8 and remained mostly uninvestigated due to a historical emphasis on major diseases, such as blast and bacterial blight9,10,11. The prevalence of RKS further increased in paddy-irrigated rice areas after the introduction of hybrid rice in the 1970 s. Today, it has worldwide distribution in all the rice-growing countries throughout the Asia, Oceania, Europe, America and Africa5,9. Every year RKS is accounted for as much as up to a 5% to 20% decrease and 40% to 60% prevalence in diseased grains6. Despite the high worldwide rice yield loss caused by T. horrida, only limited information is available about its identification, the infection route and chemical control6,12. Currently, USDA (United States Department of Agriculture) has stipulated that any rice containing more than 3% infected kernels cannot be used as parboiled rice. Producers are docked when infection level in harvested grains is above 3%7.

The potential role of China in the global rice system is immense as approximately one-third of all the rice in the world is produced and consumed in China. Rice seeds from China are also being exported to most of the countries in Southeast Asia13. However, most of rice cultivars are susceptible to RKS and any possible incursion would cause severer disruption to both the rice production and international trade of China. Furthermore, crop rotation is a common agricultural practice in the rice-growing regions of China. Thus, the presence of related Tilletia sp. can cause the ambiguity during leading to raise the trade barriers to rice export.

The current diagnostic protocol for RKS of rice involves the morphological identification of spores, followed by germination to confirm the identity of the pathogen. But these protocols are very time consuming, labor intensive and require highly skilled personnel to distinguish the morphological similar species14. Therefore, there is a dire need to develop a simple and rapid method for the detection and identification of T. horrida in rice seeds from the other common contaminant fungal species. Traditional PCR based detection methods are simple, rapid, highly specific and sensitive for the target species and can be used to discriminate allelic homologues genomic fragments with minor nucleotide differences. Some of such differences exist in the internal transcribed spacer (ITS) sequences of eukaryotes. ITS region between the nuclear small and large subunit ribosomal DNA has proved useful to distinguish the closely related species of fungi14.

This work describes the rapid and accurate PCR based method for the quarantine detection and identification of T. horrida in the rice seed samples. The partial ITS region sequences were targeted in the present study to design species specific primers. According to our knowledge this is the first report on the use of conventional PCR based identification and detection of T. horrida by using it partial ITS sequences.

Results

Primer design and PCR

To design the primer and develop the PCR assay for the detection of T. horrida, ITS rDNA sequence of T. horrida CN1 (Accession No. DQ827699.1) was used. The sequence was analyzed for homology with the target Tilletia species including T. controversa, T. walker, T. ehrhartae, T. indica and T. caries (Fig. 1). The forward and reverse primers were designed for the amplification of the target region only from T. horrida.

Figure 1
figure 1

Alignment and analysis of partial sequences of ITS region of 5.8 S rDNA of selected Tilletia species.

The red frame indicates the nucleotide differences among the six Tilletia species.

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Specificity of the designed primers

The specificity of the T. horrida primers was first evaluated using a collection of genomic DNA samples from T. horrida, T. controversa, T. walkeri, T. ehrhartae, T. indica and T. caries as template for PCR assay. The analyzing of amplicons revealed that the designed primers only amplified a 503-bp fragment from the DNA of T. horrida but not from the other five tested Tilletia species (Fig. 2). Furthermore, when the PCR was performed using genomic DNA of the 22 other fungi from the genera other than Tilletia (Table 1), a fragment of the same size could not be amplified (Fig. 3), suggesting that there was no cross species specificity for the PCR assay developed for the detection of T. horrida.

Table 1 List of fungal species, their hosts and geographical origin.
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Figure 2
figure 2

Specificity test of the primers for the target species of Tilletia.

Lane 1: negative control, Lane 2: T. horrida CN1, Lane 3: T. controversa, Lane 4: T. walker, Lane 5: T. ehrhartae, Lane 6: T. indica, Lane 7: T. caries.

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Figure 3
figure 3

Specificity test of the primers for Tilletia horrida and the other fungal genra.

Lane 1: Positive control (T. horrida CN1), Lane 2: Pyricularia grisea, Lane 3: Ustilaginoidea virens, Lane 4: Rhizoctonia solani, Lane 5: Fusarium moniliforme, Lane 6: Aspergillus flavus, Lane 7: Botrytis cinerea, Lane 8: Nigrospora sphaerica, Lane 9: Alternaria alternata, Lane 10: Botryosphaeria dothidea, Lane 11: Coniothyrium diplodiella, Lane 12: Colletotrichum gloeosporioides, Lane 13: Podosphaera leucotricha, Lane 14: Glomerella acutata, Lane 15: Pestalotiopsis theae, Lane 16: Coniella granati, Lane 17: Phomopsis fukushii, Lane 18: Sclerotinia sclerotiorum, Lane 19: Penicillium expansum, Lane 20: Monilinia fructicola, Lane 21: Gaeumannomyces graminis Lane 22: Ascochyta eriobotryae, Lane 23: Bipolaris sorokiniana, Lane 24: Negative control.

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Detection of T. horrida in rice seeds

The optimized PCR assay was performed to evaluate RKS in 21 rice seed samples, out of all these samples, 6 (28.6%) were found to be PCR positive, indicating these were contaminated with the pathogen T. horrida, while remaining 15 seed samples were free of the causative pathogen (Fig. 4). These results were also confirmed by the morphological studies of the spores isolated from all the samples under investigation. The specificity of this test was further confirmed by performing PCR using genomic DNA from 11 different strains of T. horrida (Table 1) and spores isolated from healthy and infected rice seeds. The diseased status of the seeds was confirmed prior to the experiment from AEEIQB, China. All the 11 strains of T. horrida, and infected seeds showed the positive results after agarose gel electrophoresis (Fig. 5). Whereas, no PCR product was obtained in the case of rice seeds with confirmed healthy status.

Figure 4
figure 4

Detection of Tilletia horrida in rice seeds.

Lane 1–21: Rice seed samples; Lane 22: Positive control (T. horrida CN1), Lane 23: Negative Control.

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Figure 5
figure 5

PCR assay for different strains of Tilletia horrida and rice seeds.

Lane 1 & 20: negative control, Lane 2–12: Tilletia horrida strains CN1, HUN-1, AH-1, HEB-1, HIN-1, JS-1, FJ-1, SC-1, YN1, US1, IN1, respectively, Lane 13–15: infected seed samples, Lane 16–18: healthy seed samples, Lane 19: positive control (T. horrida CN1).

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Sensitivity of the test

Firstly, the sensitivity of the present method was tested by varying the number of spores isolated from the T. horrida contaminated seeds in the PCR reaction mixture. The results depicted that the lower limit for the detection of target pathogen was 30 spores per 25 μl of PCR reaction mixture (Fig. 6). No amplified product was observed in UV illuminator when less than 30 spores were used in the reaction mixture.

Figure 6
figure 6

Sensitivity test for the detection of Tilletia horrida spores.

Lane 1–6: No. of spores (1, 10, 20, 30, 40 & 50, respectively) per 25 μl of reaction mixture; lane 7: positive control (Tilletia horrida CN1); lane 8: negative control.

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Secondly, in order to know the sensitivity of this method for the detection of T. horrida in rice seeds, artificially 10, 15, 20, 25, 30, 50 and 100 spores were added into 100 g healthy rice seeds per sample. The results indicated that the lower limit for this method was 25 spores/100 g rice seeds and no PCR product was obtained when less than 20 spores were added in the rice seeds (Fig. 7).

Figure 7
figure 7

Sensitivity test for the detection of Tilletia horrida spores in rice seeds.

Lane 1 & 11: DNA ladder; lane 2–8: spore numbers 10, 15, 20, 25, 30, 50 and 100 respectively; lane 9: positive control (Tilletia horrida CN1); lane 10: negative control.

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Thirdly, to test the sensitivity of this method at the DNA level, the purified DNA (100 ng, 10 ng, 1 ng, 100 pg, 10 pg, 1 pg and 100 fg, respectively) was used as a template for the PCR and the results indicated that this method could detect ≥100 pg genomic DNA of T. horrida (Fig. 8).

Figure 8
figure 8

Sensitivity test for the detection of the DNA of Tilletia horrida.

Lane 1 & 11: DNA ladder; lane 2: positive control; lane 3–9: template DNA concentrations (100 ng, 10 ng, 1 ng, 100 pg, 10 pg, 1 pg and 100 fg, respectively); lane 10: negative control.

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Discussion

The emerging epidemic trends throughout the world has placed a greater stress to manage rice kernel smut in the future9. Thus, rapid detection and identification of T. horrida in rice is crucial for the implementation of surveillance and quarantine regulation in the international rice trade. Detection assays using classical PCR techniques have been developed for numerous plant pathogens, including bacteria, viruses, and fungi15. These tests are attractive for several reasons. First, the assays are extremely sensitive and highly specific for the pathogen in question. Second, PCR tests require minimal amounts of sample material, and commercial kits are available for extracting high quality genomic DNA from a wide variety of organisms. Finally, PCR reactions are relatively simple to set up and perform, and results can be obtained quickly, usually within a few hours. In this study, a simple method for the rapid detection and identification of T. horrida in rice seeds was developed by the analysis of the ITS region of rDNA from the six Tilletia species. The ITS region is long tandem DNA repeat array that is located between ITS1 and ITS2 rRNA genes in the (rDNA) unit in eukaryotes16. This region has a high degree of variation even between the closely related species and is widely used for the molecular phylogeny and taxonomic studies. According to a previous study, the ITS region has the highest probability of successful identification for the broadest range of fungi, with the most clearly defined barcode gap between inter- and intraspecific variation among the regions of the ribosomal cistron. ITS will be formally proposed for adoption as the primary fungal barcode marker to the Consortium for the Barcode of Life, with the possibility that supplementary barcodes may be developed for particular narrowly circumscribed taxonomic groups17. Other than belonging to the same genus, T. horrida is not closely related to the other species included in the study, they possess considerable genomic differences to differentiate them from each other (Fig. 1). Therefore, to distinguish T. horrida from other five Tilletia species, PCR method was developed by designing a specific pair of primers to amplify a 503 bp fragment of T. horrida ITS1 + 5.8 S + ITS2 rDNA region. The designed primers did not amplify any product from the genomic DNA of any of the tested fungi, except T. horrida implicating the specificity of the developed PCR protocol. Many other workers have also reported the use of ITS regions to resolve Tilletia species comprising closely related T. horrida, T. walker, and T. indica, T. contraversa, T. laevis, T. caries, T. bromi and T. fusca14.

In the last two decades, isozyme analysis was used to distinguish the isolates of T. indica from T. horrida by using proteins extracted from germinated teliospores. However, considerable experience with the interpretation of complex isozyme polymorphisms associated with Tilletia species is required, and this is not considered as a practical approach for the routine identification18,19,20. Several studies have been reported for the identification and differentiation of T. indica (a quarantine pathogen causing Karnal bunt of wheat) from T. walkeri and T. horrida20,21,22 by using conventional PCR with. In 2006, a molecular protocol, using quenched fluorescence resonance energy transfer (FRET) probes, was developed for the detection and differentiation of T. indica from T. walkeri, T. horrida and T. tritici23. A one-tube fluorescent assay for the quarantine detection and identification of T. indica has also been developed14. However, all these reports just focused on the detection of T. indica and not to distinguish T. horrida from the other Tilletia species. Moreover, despite of the high sensitivity and accuracy, these methods cannot be commonly used because of the high-priced apparatus and reagent kits. Similarly, other methods such as enzyme-linked immunosorbent assay (ELISA)24, sequence-characterized amplified region (SCAR)25 inter simple sequence repeat (ISSR) marker26 have also been developed for the diagnosis of T. caries and T. controversa, respectively. However, these methods are either expensive or time-consuming.

Compared to these methods, the method for detection and differentiation of T. horrida from other Tilletia species in our study possesses numerous advantages. It depends on common techniques of molecular biology and needs merely basic PCR reagents, electrophoresis apparatus and thermocycler. This equipment is relatively cheap, inexpensive to maintain, and simple to manipulate. In our method we report the direct use of teliospores for the diagnosis of T. horrida without the culturing and genomic DNA isolation. Thus eliminating the lengthy culturing and difficult morphological identification procedures27,28. Furthermore, sensitivity test results showed that the present method can be employed using only ~30 spores in the reaction mixture. The traditional morphological identification methods usually require considerable expertise and a significant of ~50 spores for statistical determination14,29. The potential benefits of this technology can be especially recognized in the regulatory field, where both the timeliness and accuracy of identifications are crucial20,30.

In our study, the time required for this detection is less than three hours, therefore, this developed technique can be used to screen out a large number of samples in a short time. Hence, it will be extremely useful to resolve the disputes regarding contamination of rice with smut teliospores. In this study, we have also confirmed the identity of the pathogen of the kernel smut of rice using the specifically designed primers.

This information would be useful in establishing quarantine areas and preventing the contamination of clean rice shipments with teliospores from infested grains.

Methods

Fungal strains

All the fungal strains (Table 1) used in the present study were provided by the Anhui Entry-Exit Inspection and Quarantine Bureau (AEEIQB), China. The fungal cultures were maintained on potato dextrose agar (PDA) medium and stored at 4 °C.

Isolation of T. horrida spores

The rice seed samples used for the detection and isolation of T. horrida spores were also supplied by the courtesy of AEEIQB, China that were collected from the different geographic locations (Table 1). The rice seeds were surface-sterilized in 0.1% sodium hypochlorite for 5 min, rinsed in sterile distilled water for 30 sec twice, striped and the teliospores scraped into a clear tube standby. The isolated spores were identified and confirmed by AEEIQB, China using Chinese National Standard31 for the identification of T. horrida. The spore counting was performed using hemocytometer.

Extraction of genomic DNA

Genomic DNA of T. horrida was extracted directly from the isolated spores using the Fungal DNA Kit (Omega Bio-Tek) according to the manufacturer’s protocol. Whereas, the genomic DNAs of the other tested pathogens (Table 1) were extracted from 50 mg mycelia using the same kit. The spores and mycelia were treated with a minibeadbeater (607EUR, Biospec, USA) before isolation of the DNA. The DNA concentration for each sample was determined by Nanodrop (NanoVue Plus, GE Healthcare Life Sciences).

Primer designing and PCR amplification

To design the primers, Internal Transcribed Spacer (ITS) sequence of ribosomal DNA of T. horrida, Accession No. DQ827699.132 was retrieved from the NCBI database. The sequence was compared and analyzed with the other Tilletia species sequences from the NCBI database using online tools BLASTN33 and Clustal Omega34. The forward primer (HF-F): 5′-GAGAGTCAACTTATGTTCA-3′ and reverse primer (HF-R): 5′-GATGAAAGTTACTCTCAT-3′ were designed by using the Bioedit software (v7.0.5) to amplify 503 bp fragment of 5.8 S ribosomal DNA (rDNA) of T. horrida. The PCR conditions were optimized using T. horrida CN1 as template DNA. The DNA amplification was carried out in DNA Engine System PT-200 (Bio-Rad) using 25 μl reaction mixture containing 1 U ExTaq polymerase, 2.5 μl of 10× ExTaq buffer, 2 μl Mg2+ (25 mM), 200 μm of dNTPs, 1 μm of each primer and DNA template (50 ng). The thermal cycler was programed for one cycle of initial denaturation of 5 min at 94 °C; followed by 30 cycles of 1 min at 94 °C, 30 sec at 50 °C and 30 sec at 72 °C with a final extension for 5 min at 72 °C; and a holding temperature of 4 °C. A negative control, replacing the DNA template with sterile distilled water and DNA ladder DL2000 were also run in parallel. The PCR products were electrophoresed using 1% agarose gel in 1xTAE buffer for 45 min at 90 V to analyze the results.

Specificity of the test

To determine the specificity of the PCR and primers to T. horria, the PCR was carried out with different species of Tilletia and other common contaminant fungal strains (Table 1) using the above mentioned optimized PCR conditions.

Detection of T. horrida in rice seeds

A total of 21 rice seed samples, provided by AEEIQB, China, were used for the detection of T. horrida. For each sample, 10 g of the seeds were washed with sterile distilled water for 30 sec twice and saturated in sterile distilled water for 5 min. The saturated water was centrifuged at 12000 rpm for 10 min, then discarded the supernatant and re-suspended the pellet in 50 μl water. 10 μl of this spore suspension was directly used as template DNA for the PCR.

Sensitivity of the test

Firstly, the sensitivity test for the detection T. horrida contaminated seeds was performed by varying the number of spores per 25 μl of PCR reaction mixture. PCR was performed using individual reaction mixtures containing 1, 10, 20, 30, 40 & 50 spores of T. horrida, respectively, followed by agarose gel electrophoresis analysis.

Secondly, artificially 10, 15, 20, 25, 30, 50 and 100 spores were added into the individual 100 g healthy rice seeds samples and then, the spores were collected by seed washing and centrifugation. The PCR was performed using the individual spore suspensions.

Thirdly, in order to know the sensitivity of this PCR method for DNA limit, the purified DNA (100 ng, 10 ng, 1 ng, 100 pg, 10 pg, 1 pg and 100 fg, respectively) was used as a template in the indicidual PCR assays followed by the same cycling conditions as described above.

Additional Information

How to cite this article: Chen, Y. et al. Simple and rapid detection of Tilletia horrida causing rice kernel smut in rice seeds. Sci. Rep. 6, 33258; doi: 10.1038/srep33258 (2016).