Development of a nested-PCR assay for the rapid detection of Pilidiella granati in pomegranate fruit

Pilidiella granati, a causal agent of twig blight and crown rot of pomegranate, is an emerging threat that may cause severe risk to the pomegranate industry in the future. Development of a rapid assay for the timely and accurate detection of P. granati will be helpful in the active surveillance and management of the disease caused by this pathogen. In this study, a nested PCR method was established for the detection of P. granati. Comparative analysis of genetic diversity within 5.8S rDNA internal transcribed spacer (ITS) sequences of P. granati and 21 other selected fungal species was performed to design species-specific primers (S1 and S2). This primer pair successfully amplified a 450 bp product exclusively from the genomic DNA of P. granati. The developed method can detect 10 pg genomic DNA of the pathogen in about 6 h. This technique was successfully applied to detect the natural infection of P. granati in the pomegranate fruit. The designed protocol is rapid and precise with a high degree of sensitivity.

China was ranked first in the world with 1.2 million tons annual production of pomegranate and total planting area about 120,000 hm 2 in 2012 17 . P. granati has caused substantial economic loss to pomegranate industry in a number of countries including China 7 . We have previously reported P. granati as a casual agent of twig dieback and fruit rot with 10 and 30% disease incidence in the major pomegranate cultivation area of China 14 . The pathogen reduced both the quality and yield of pomegranate. Therefore, it is necessary to develop a rapid and accurate method for the detection of P. granati that can be implemented for the routine diagnosis and management of the pathogen.
Traditional fungal identification protocols include isolation, culturing and studying the morphological characters combined with physiological tests. These methods are labor intensive, time-consuming. Moreover, highly skilled and experienced personnel are required to identify less commonly encountered pathogens and variant strains 18,19 . However, with the advancement in the molecular biology, authentic DNA barcodes are available as a powerful tool for the identification of fungal species. One of the commonly used markers is highly repetitive internal transcribed spacer (ITS) sequences within the ribosomal RNA gene cluster. The success of these sequences along with PCR has eliminated the use of even more correct fungal protein-coding DNA sequences [18][19][20][21][22] .
PCR-based diagnostic methods are well documented for numerous plant pathogens, including bacteria, viruses, and fungi [23][24][25] . These methods are rapid, sensitive and highly specific 26 . Therefore, in present work, nested PCR technique has been used for the rapid and accurate detection of P. granati in pomegranate. Furthermore, this is the first report on the PCR-based approach to detect P. granati.

Results
Primer design and nested PCR. In the present work, the nested PCR method has been developed for the detection of P. granati in the pomegranate fruit. In order to design the specific primers, ITS sequence of 5.8S rDNA of P. granati (GenBank accession No. KF560320.1) was used (Fig. 1). The target sequence was compared with 5.8S ITS regions of seven other fungal strains (Table 1) using BioEdit v7.0.5 software. The aligned sequences were used to design the S1 and S2 primers (Fig. 2). In the first round of amplification, universal primer pair ITS1 ⁄ ITS4 was used. Whereas, in the second round of amplification, a predicted 450-bp DNA fragment was successfully amplified using S1 and S2 primers.
Specificity of the assay. The specificity of the primers was tested by using genomic DNAs of 21 different fungal pathogens (Table 2). An expected 450 bp DNA fragment was amplified using the S1/S2 primers only from P. granati. No PCR products were obtained from the other tested fungal strains (Fig. 3). The specificity was further tested by using the genomic DNA of five other fungal pathogens of pomegranate (Glomerella cingulate, Penicillium purpurogenum, Monochaetia pachyspora, Cercospora punicae and Sphaceloma punicae). Again, no PCR products were obtained with these pomegranate pathogens (Fig. 4). The amplification of PCR product exclusively from the genomic DNA of P. granati indicated that the designed primers were especially specific for the target pathogen.
Sensitivity of the assay. The sensitivity of the designed protocol was tested by using different concentrations of genomic DNA of P. granati as a template in the individual nested PCR assays. In the first step, the conventional PCR reaction was carried out using S1 and S2 primers. The PCR product analysis indicated that the     Table 2. List of fungal species and their hosts used to test primer specificity.
lower limit for the detection of target pathogen was 10 ng of DNA per 25 μ l of PCR mixture (Fig. 5). To increase sensitivity, the nested PCR protocol was performed using a universal primer pair (ITS1 and ITS4) and a primary PCR primer pair (S1 and S2). This enhanced the sensitivity of the assay and the detection of the pathogen with 10 pg of DNA was obtained (Fig. 6). Thus, nested PCR increased the lower detection limit of genomic DNA from 10 ng to 10 pg.
Detection of P. granati in pomegranate fruit. The nested PCR was performed to diagnose the P. granati infection in the pomegranate samples that were collected from the different areas of Anhui Province, China. To validate the protocol, artificially infected pomegranate fruits were also used. The genomic DNAs were isolated from naturally infected, artificially infected and healthy control fruits and subjected to the nested PCR assay. Both the naturally infected and artificially infected samples were found to be positive for P. granati as a 450-bp PCR   product was obtained on the agarose gel. Whereas, no PCR products were obtained with DNA from the control samples (Fig. 7).

Discussion
The disease caused by P. granati, is an emerging threat to the rapidly expanding pomegranate industry in many regions of the world. It has been reported to cause crown rot, dry rot and dieback twig blight of pomegranate in many countries including Eastern Mediterranean, Turkey, India, Greece, Cyprus, and China 7,9,12,14-16 . A comprehensive survey in Greece showed that disease incidence was 29 and 50% of pomegranate fruit rot by P. granati at various locations in 2011 & 2012, respectively that increased to 34-53% in all the commercial pomegranate orchards in 2014. Pycnidia of the pathogen were found in 77% of the mummified fruits, 25% of the blighted shoot and 19% of the crown of trees with symptoms of rots that were left in the orchard. Moreover, the disease incidence was higher in the areas where dark brown to black fruit mummies were seen scattered on the orchard floor 7 . In a few countries, the pomegranate disease caused by P. granati has already acquired the status of quarantine disease. In 2006, all the grafting material that imported from India was destroyed after the diagnosis of C. granati in Israel 15 .
To develop active surveillance and management of dry rot in pomegranate industry is critical for avoiding the yield losses by P. granati. 27 . A rapid and precise detection of P. granati is a preliminary step to achieve this goal. However, traditonal identification appraoch involves the identification based on culturing and morphology, which is time consuming 18 .
Molecular-based methods such as PCR have greatly improved the detection of microbes present in the environment 28 . PCR based assays are more rapid, sensitive, specific and accurate and have been often implemented for the routine diagnostics of a variety of pathogens 24,25,[29][30][31][32][33] . In the present work, we have used nested PCR as a rapid approach for the detection of P. granati. Analysis of ITS sequences of rDNA of P. granati and seven other fungal strains was performed to design primary PCR primer pair. The developed protocol was successfully used for the exclusive amplification of the 450 bp fragment from P. granati genomic DNA. Thus, this method can discriminate P. granati from all the other fungi tested. In the consortia of the barcodes of life, ITS sequences of nuclear rDNA serve as universal DNA barcodes. These loci have become very attractive alternatives to the traditional protocols mainly due to the development of successful PCR and sequencing methods. Even though the ITS sequences can be readily amplified by universal ITS primers, there is still sufficient interspecific sequence divergence. This diversity within ITS region can be exploited for the species identification by using carefully designed species-specific primers 18,22,24,34 . Therefore, in the present work ITS region of the P. granati was used to develop the detection protocol.  The primer with high specificity in the PCR based diagnostics is of prime importance. Therefore, 21 different fungal strains, including P. diplodiella were used to test the specificity of the S1/S2 primer pair. In the second round of amplification, no PCR products were obtained with any of the tested strains. Only P. granati gave the positive results. The specificity of the designed primers was also tested for the seven different pomegranate pathogens. However, again, no PCR products were obtained with any of these pathogens. Thus, these results indicate that the developed protocol is specific for the P. granati. The primers (S1 and S2) designed in the present nested PCR protocol are not claimed to be highly species specific. Even though, when the designed primer pair was used to detect P. diplodiella, no PCR products were obtained. We did not aim to make the primers highly species specific because no other Pilidiella species have been reported to infect pomegranate plant. P. granati is host specific and the sole pathogen of the pomegranate from the genus Pilidiella. When it infects the pomegranate, it penetrates inside the host tissues. Thus, host tissues might be used for detection of the pathogen. Moreover, in the developed protocol, the samples were surface sterilized before the extraction of fungal genomic DNA. Consequently, the probability of the presence of any other Pilidiella species as a contaminant inside the fruit tissues is very rare. Therefore, no further work was carried out to analyze and improve the species-specificity.
Although the conventional PCR is considered to be the most suitable diagnostic technique for the detection of various kinds of pathogens. It has certain detection limit when the target DNA concentration is low. It is very often necessary to enhance the sensitivity of the reaction. Several PCR techniques, notably including nested PCR, qPCR, Bio-PCR and co-operational PCR coupled with dot blot hybridization, have been developed to increase the sensitivity of the PCR based assays. Among these, nested PCR is the most frequently used method to obtain the acceptable level of sensitivity 19,24,28,[35][36][37] . The earlier infection of P. granati in the pomegranate plants and young fruits is either latent or too low to be detected. In the present work, when conventional PCR was used, the lower detection limit for template DNA was 10 ng. The nested PCR technique was used to enhance the sensitivity of the PCR assay. This increased the sensitivity of the assay and detection of the pathogen was possible when as low as 10 pg of P. granati DNA was present. Many other researchers have used nested PCR to increase the sensitivity of the reaction for the detection of pathogens 19,24,[37][38][39][40][41][42] .
To validate the current protocol, healthy pomegranate fruits were artificially inoculated with P. granati followed by the detection of pathogen. The genomic DNA was extracted from the artificially inoculated, naturally infected and control healthy samples followed by detection of the pathogen by nested PCR approach. The results showed that the developed protocol successfully detected the P. granati infection only in both the naturally and artificially infected pomegranate fruit in 6 h. No PCR products were obtained in healthy samples. Thus, these results indicate that method developed in the present work is rapid, accurate and highly sensitive. It is a promising and alternative method to the traditional diagnostic and identification protocols for the detection of P. granati. This method will be useful for the early detection of P. granati infection. The technique will be helpful, especially for the farmers to manage the disease in time. Furthermore, this method can also be applied to study the epidemic trends of this disease in the pomegranate cultivation regions.

Methodology
Fungal strains. All the fungal strains used in this work were isolated from the different fruits. These fruits were collected from the different areas of Anhui Province, China. These fungal cultures were maintained on the potato dextrose agar (PDA) medium and stored at 4 °C. The isolates were firstly identified by cultural and morphological characters. The identity of these strains was further confirmed by PCR using ITS1 and ITS4 universal primers followed by standard sequencing. The sequences were used to identify the isolates by using the online bioinformatic tool BLASTN 43 . Extraction of fungal genomic DNA. Fungal strains were grown on the individual PDA plates at 28 °C for 48-72 h. The fungal mycelial mass (50 mg) from each strain was used to extract genomic DNA using the Fungal DNA Kit (Omega Bio-Tek). The isolation was carried out according to the manufacturer's protocol. DNA concentration for each sample was measured by using NanDrop UV spectrophotometer (NanoVue Plus, GE Healthcare Life Sciences).
Nested PCR. First round of nested PCR was carried out using universal primers ITS1 (5'-TCCGTAGGT GAACCTGCGG-3′ ) and ITS4 (5′ -TCCTCCGCTTATTGATATGC-3′ ) 44 . The amplification was performed in PCR tube containing 10X Taq buffer (2.5 μ l), 25 mM MgCl 2 (2.0 μ L), 0.8 mM dNTP, 0.4 μ m of each of ITS1 and ITS4 primers, 5 U Taq DNA polymerase and 50 ng template DNA. The final volume of the reaction mixture was made up to 25 μ L with sterile distilled water. The optimized thermocycler conditions for the reaction were initial denaturation at 94 °C for 5 min, 35 cycles at 94 °C for 30 s, 58 °C for 30 s, 72 °C for 30 s and final extension at 72 °C for 10 min. The second round of amplification was carried out using same final concentration of the reagents as described above, except replacing the DNA template with 0.5 μ l PCR product from the first round of amplification. The thermocycler conditions were also kept the same except that the annealing temperature was reduced to 52 °C. The PCR products were checked using 1% agarose gel with DNA ladder DL2000.
Specificity of the assay. Specificity of the S1 and S2 primer pair for the detection of P. granati was determined by using the genomic DNAs isolated from P. granati and 21 different fungal species ( DNAs isolated from these strains were used as template for the nested PCR assay as described above. To confirm the specificity of the primers for different pomegranate pathogens, the nested PCR assay was carried out using the seven common pomegranate pathogens including Glomerella cingulate, Penicillium purpurogenum, Botrytis cinerea, Aspergillus niger, Alternaria spp., Trichoderma spp., Pestalotia brevista. Sensitivity of the assay. The sensitivity of the nested PCR for the detection of P. granati was determined by using the different concentrations (1.0 ng-100 fg) of genomic DNA as template.
Detection of P. granati in the infected fruits. The healthy and infected fruit samples were collected from the different orchards of Huaiyuan County, Anhui, China in sterile polythene bags and stored at 4 °C in laboratory conditions. The artificially infected samples were prepared by inoculating the healthy fruits with P. granati 14 . The genomic DNA was isolated from the artificially inoculated, naturally infected and healthy (control) pomegranate samples by using the standard protocol 45 with minor modification. The surface of each sample was disinfected with 75% ethanol for 1 min and washed with sterile water twice. About 50 mg of each fresh fruit tissues was individually grounded in liquid nitrogen with a twister in a 1.5 mL Eppendorf tube. After that 900 μ l CTAB extraction buffer and 90 μ l SDS (10%, w/v) were added to the each tube and vortexed. The tubes were incubated at 60 °C for 1 h. The genomic DNA was extracted from the supernatant with phenol/trichloromethane/isoamyl alcohol mixture (25:24:1) followed by precipitation with equal volume of isopropanol. The pellet was washed twice with 70% ethanol. The pellet was air dried and dissolved in 70 μ l TE buffer. The DNA concentration of each sample was estimated by the Nanodrop UV spectrophotometer (NanoVue Plus, GE Healthcare Life Sciences). The nested PCR was performed as described above. The genomic DNA from the P. granati was used as positive control in all the experiments. In negative control, genomic DNA was replaced with sterile distilled water.