Development of a Multiplex PCR Method to Detect Fungal Pathogens for Quarantine on Exported Cacti

Most Cactaceae are native to the Americas, including north, central, and south America. Cacti are distributed throughout a broad range of climatic conditions, including xeric deserts of the American southwest to rain forests of South America (Cruz et al., 1997), resulting in a morphologically heterogeneous group that is classified in three subfamilies (Cactoideae, Opuntioideae, and Pereskioideae) composed of approximately 100 genera and over 1,500 species. Recently, cactus farms in Korea, Japan, and China have started grafting cacti. By grafting two different cactus species, including photosynthetic stocks and non-photosynthetic scions are grafted and form decorative colored scions. Currently, grafted-cacti products from Korea exceed 70% of the world’s cacti trading market due to the beauty in cacti appearance and variability in shape and color (Song et al., 2009a, 2009b). The grafted-cacti are cultivated under greenhouse conditions at warm temperatures and high humidity during the entire growing season. Therefore cacti frequently were infected by a variety of fungal diseases (Chang et al., 1998; Choi et al., 2010; Hyun et al., 1998; Kim et al., 2000; Kim et al., 2007). Cactus diseases are a major factor in greenhouse grafted-cacti, particularly stem rot caused by F. oxysporum and B. cactivora (=Helminthosporium cactovorum Petr., (1931) and =Drechslera cactivora) (Petr.) M.B. Ellis, (1971) (Chang et al., 1998; Hyun et al., 1998). Other diseases, anthracnose (Collectotrichum spp.) are also a problem and found worldwide as cactus late blight disease (P. cactorum and P. nicotianae). In 1999 and 2000, a major stem rot disease outbreak was observed in the Suwon regions (National Horticulture Research Institute) and other primary grafted-cacti producing districts, such as Anseong, Eumseong, and Goyang. Stem rot symptoms caused by Alternaria sp., Fusarium sp., and B. cactivora were frequently identified in cacti greenhouses (Kim et al., 2000). Imported counties, including Europe, Israel, and Southern Asia classified the cacti pathogens, that are F. oxysporum, B. cactivora, P. cactorum and P. nicotianae as quarantine pathogens. Therefore, disease or contamination free certification is now required before plants can be imported.

Disease and pathogen diagnosis can be time consuming and sophisticated processes, because most cactus pathogens produce similar disease symptoms. Therefore, there has been an increased demand for rapid diagnosis methods. Polymerase Chain Reaction (PCR)-based detection method has been adopted as the most reliable and rapid technique in the scientific community (Hameed et al., 2014; Kwak et al., 2014). Specific primer sets were developed with fungi unique gene sequences to detect fungal pathogens (Buchman et al., 1990; Kan et al., 1993), Multiplex PCR for pathogen diagnosis, permits simultaneous amplification of several pathogens in a single reaction mixture, and facilitates cost-effective diagnosis (Aguilar et al., 2000; Corias et al., 2003; Fan et al., 1998; Grondahl et al., 1999; Liolios et al., 2001). The objective of this study was to develop a PCR detection method that amplified highly conserved target gene sequences, analyze the detection sensitivity, and the primer specificity to cactus pathogens. Results of this study served to provide a cost effective and rapid method to prove grafted-cacti are disease free for the required certification to export grafted-cacti from Korea.

Four quarantine fungal pathogens were obtained from the Korean Agriculture Culture Collection (KACC), including the standard isolates B. cactivora (#40851), F. oxysporum (#44306), P. cactivorum (#40174), and P. nicotianae (#40403). All isolates used in this study were confirmed by molecular and morphological characteristics (data not shown). All quarantine pathogens cause cactus root or stem rot diseases. Quarantine fungal isolates were cultivated using standard cultivation methods. A pure culture of each fungal isolate was inoculated on Potato Dextrose Agar (PDA: B. cactivora, F. oxysporum) or 10% V8 Juice Agar (VJA: P. cactivorum, P. nicotianae) and subsequently incubated at 27°C for 5 days. Following incubation, the spore suspension concentration was adjusted from 1 × 10⁹ to 1 × 10¹ spore/ml using a hemacytometer. A syringe was used to inoculate fungal spore suspension onto the cactus stems in decreasing concentrations. Grafted cactus stems inoculated with fungal spore suspensions were incubated at 25°C for 5 days.

For DNA extraction, B. cactivora and F. oxysporum hyphae were grown in PDB at 27°C for 5 days at 27°C. P. nicotianae and P. cactorum were incubated in 10% V8 juice broth. The hyphae were harvested by centrifugation (5,000 rpm for 5 min). The CTAB method was used to extract genomic DNA from the cultured quarantine fungi (Lee and Taylor, 1990). The extracted DNAs were dissolved in distilled water. Cacti genomic DNA, which was injected with the fungal pathogen, were extracted (100–200 mg frozen weight) using the Qiagen DNeasy Plant Mini Kit. Species-specific primer pairs, including Fo.F-Fo.R (F. oxysporum), BC.F-BC.R (B. cactivora), PN.F-PN.R (P. cactorum), and PC.F-PC.R (P. nicotianae) were designed based on specific sequence data for the internal transcribed spacer (ITS) region and the Ypt1 gene in GenBank. P. cactivora specific primers PN.F and PN.R were previously described (Li et al., 2013). The following reaction mixture was used for PCR with individual primer pairs: l ul (40 ng) of diluted template genomic DNA, 1 ul 10 mM (dNTP), 2 ul of 10× Reaction buffer, 1 ul (10 pmole) primer, and 1 unit of Taq DNA polymerase (Bioneer, Korea) in a total volume of 20 ul. PCR amplification conditions included 5 min of denaturation at 94°C; followed by 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 15s; and a final extension step of 72°C for 10 min. Each multiplex contained four primer pairs designed to produce amplicons sufficiently different in size and migration rate to identify the four pathogen species (B. cactivora, F. oxysporum, P. cactivora, and P. nicotianae). Primer information is presented in Table 1. Reaction mixture reagents were the same as described above for single-primer-pair PCR. The PCR tubes were maintained on ice and PCRs were performed in a PTC-100 programmable Thermal Controller (MJ Research, INC. USA). All PCRs were run with the same cycling program used for single-primer-pair PCR. A 4 ul sample of product from each PCR was electrophoresed in a 1.3% agarose gel with 0.3 ug/ml of ethidium bromide for 15 to 20 min. DNA bands were visualized and UV documented with a BioDoc-IT 220 imaging System 3 Door/8.0 LCD With M-20 Transilluminator (Analytik Jena AG, Germany).

The four pairs of species-specific primers were frequently amplified and detected the quarantine pathogens (Table 1). The PCR assay was primarily established as a monospecific assay with individual genomic DNA of the four fungal pathogens. Standard PCR sensitivity was generated with PCR product values obtained and used to optimize PCR efficiency. All tested quarantine pathogens were successfully amplified and visualized by ethidium bromide stained gel analysis (data not shown). More accurately, fungal pathogens (B. cactivora, F. oxysporum, P. cactorum, P. nicotianae) for the species-specific region were also successfully amplified with the defined primer pair (data not present). Sensitivity of the species-specific primers was evaluated with serial diluted DNA concentrations from 100 to 0.01 ng. Under in vitro conditions, the species-specific fungal primers, F.oxF-F.oxR, B.CF-B.CR, and P.NF-P.NR were detectable at 0.01 ng of genomic DNA; and the P.CF-P.CR primer produced an amplicon of the appropriate size in a 1 ng concentration of genomic DNA (Fig. 1). The four different fungal pathogens were detected using 100 ng of DNA by multiplex PCR following ethidium bromide staining. For multiplex PCR, the four primer sets were combined in a single tube to simultaneously identify four fungal pathogens. B. cactivora and F. oxysporum were detected in the agarose gel at a sensitivity level of only 0.01 ng. P. nicotianae sensitivity was 0.1 ng of DNA and 1 ng of DNA for P. cactorum (Fig. 2). The sensitivity of the multiplex PCR was comparable to single-primer-set PCR sensitivity. These results indicated up to three suspected pathogens can be identified in a single PCR.

Cacti were inoculated with spore suspensions (10^1–9) for detection pathogen from symptomatic cactus. The most of efficiency demonstrated in 10^7–9 experiments with strains of each of the four fungal pathogens (Fig. 3). Quarantine pathogens inoculated cacti were incubated at room temperature (25°C) for 5 days. All pathogens caused typical disease symptoms in cactus species with spore density above 10⁷ cfu/ml. Following sampling from the inoculated site, genomic DNA was extracted to test specificity and sensitivity of the multiplex PCR under in vivo conditions. Multiplex PCRs generated specific amplicons of correct size when templates from corresponding pathogenic fungal species were present. All multiplex PCR conditions were the same as described above. PCR reactions were positive when pathogens caused disease symptoms at 10⁷ cfu/ml, with the exception of F. oxysporum, which was detected at 10⁶ cfu/ml. This sensitivity was achieved only for the quarantine cactus pathogen samples tested using multiplex PCR (data not shown).

For test detection sensitivity of target pathogens with asymptomatic cacti, spore suspension concentration was adjusted from 1 × 10¹ to 1 × 10⁹ spore/ml using a hemacytometer. After dropping fungal spore-stock on cacti stems, inoculated cacti were dried in clean bench for 2 hrs. Inoculated cacti stems were rubbed by a cotton swab. The cotton swab put in 1.5-ml E-tube containing 900 ul distilled water for genomic DNA extraction and multiplex PCR. The multiplex PCR set showed high specificity in four different inoculated spore suspensions on asymptomatic cacti. In this case of P. nicotianae, 313 bp fragment was only generated from 10³ to 10⁹ cfu/ml with asymptomatic cacti (Fig. 4A). F. oxysporum and P. cactorum showed 210 bp, 447 bp from 10⁹ to 10⁴ respectively. B. cactivora, where the 510 base-pair appears 10⁵ to 10⁹ in treated cactus.

Our results demonstrated sensitivity similar to that of export grafted cacti cultivation contaminations with a markedly greater accuracy in diagnosis. This method is a valuable addition to the cacti grafting industry, and offers powerful diagnostic tools for the detection and identification of fungal quarantine pathogens.