Evaluation of Some Specific Primer Sets Development for Detecting Fusarium Oxysporum F. SP. Cubense Tropic Race 4 (Foc TR4) Originating From Indonesia

Fusarium oxysporum f. sp. cubense tropic race 4 (Foc TR4) strain which belong to Vegetative Compatibility Group (VCG) 01213 is the most devastating disease in global banana production. Validation of specific primer sets using the positive control (Foc TR4). In total, 50 isolates of Foc are collected from several banana production regions in Indonesia represent the group of VCG, races, genotype, cultivars, which are confirmed as Foc based on the tested using FocEf3 primer set, except Cjr-2 and Lmp-4 isolates. Foc-1/Foc-2 could amplify 34 Foc isolates included in Foc race 4. Three specific primer sets i.e. TR4-F/TR4-R, Six-1c, and TR4-F2/TR4-R1 are used to classify Foc isolates into Foc tropic race 4. TR4-F/TR4-R is known have the highest specificity as it could amplify 35 Foc isolates including positive controls (Foc TR4) compared to the other primer sets (Six-1c and TR4-F2/TR4-R1). This research indicates that there are a large number of diversity strains found in Foc isolates to be studied for further research. Race 4 of Foc (STR4 or TR4) is known to be widespread in several regions in Indonesia. Therefore, specific primer set development needs to be done to detect Foc TR4 and the most damaging strains on Foc TR4 based on molecular data.IntisariFusarium oxysporum f. sp. cubense ras 4 tropika (Foc TR4) yang termasuk ke dalam kelompok VCG 01213 merupakan patogen yang paling merusak dalam produksi tanaman pisang secara global. Validasi primer spesifik berbasis PCR menggunakan kontrol positif (Foc TR4). Total, 50 isolat Foc dikoleksi dari beberapa daerah produksi pisang di Indonesia mewakili VCG, ras, genotipe dan kultivar yang dikonfirmasi sebagai isolat Foc berdasarkan pengujian menggunakan primer FocEf3, kecuali isolat Cjr-2 dan Lmp-4. Foc-1/Foc-2 dapat mengamplifikasi 34 isolat Foc yang termasuk ke dalam Foc ras 4. Selanjutnya tiga pasang primer spesifik yaitu TR4-F/TR4-R, Six-1c, dan TR4-F2/TR4-R1 digunakan untuk mengelompokkan isolat-isolat tersebut ke dalam isolat Foc ras 4 tropika. TR4-F/TR4-R diketahui memiliki spesifitas tertinggi karena dapat mengamplifikasi sebanyak 35 isolat Foc termasuk kontrol positif (Foc TR4) dibandingkan dengan primer lainnya (Six-1c dan TR4-F2/TR4-R1). Penelitian ini menunjukkan bahwa terdapat sejumlah besar keragaman strain yang terlihat pada isolat-isolat Foc tersebut untuk dapat dipelajari lebih lanjut. Ras 4 dari Foc (STR4 atau TR4) diketahui tersebar luas pada beberapa daerah di Indonesia. Oleh karena itu, perlu dilakukan pengembangan primer spesifik untuk mendeteksi Foc TR4 dan strain yang paling merusak pada Foc TR4 berdasarkan data molekuler.


INTRODUCTION
Fusarium wilt or Panama disease caused by Fusarium oxysporum f. sp. cubense (Foc) (E.F. Smith) Snyder and Hansen is the most destructive pathogen of banana in the world. Four races of Foc have been reported based on their pathogenicity to different banana cultivars, except race 3 which is not a pathogen of banana as it only infects Heliconia spp. Race 1 infects Gros Michel (AAA), Silk and Pome varieties (AAB). Race 2 causes disease in cooking banana cultivars such as Bluggoe (ABB) (Ploetz, 2006). Race 1 of Foc gained prominence when it almost destroyed Gros Michel bananas, in Central America in the first half of the 1900s. As a result, Gros Michel cultivar is replaced by resistant Cavendish cultivar (AAA) in the early 1960s (Stover, 1962). Unfortunately, a new race of Foc, that is, race 4 is the most recently evolved and most virulent strain infecting the Cavendish cultivar as well as both race 1 and race 2 susceptible cultivars (Stover, 1972). Hence, fusarium wilt continues to be a constraint to susceptible varieties and is still considered a major threat to banana production because it cannot be controlled with fungicides.
Race 4 of Foc is first designated in Taiwan by Su in 1977 on the basis of wilt in Cavendish cultivar (Su et al., 1986). Before 1990, isolates that are classified as race 4 only caused serious losses in Cavendish genotypes in sub-tropic regions of Australia, the Canary Islands and Taiwan (Ploetz, 1990). Since then, a new variant that severely affects Cavendish cultivar in the tropics is identified. Thus, two types of Foc race 4, subtropic race 4 (STR4) and tropic race 4 (TR4) are designated. However, while Foc STR4 isolates cause disease in Cavendish cultivar in the subtropics, mainly when plants are exposed to abiotic stress, Foc TR4 isolates are pathogenic under both tropic and subtropic conditions (Buddenhagen, 2009).
Since its appearance, Foc TR4 has caused severe damage to Cavendish cultivars in TR4 had been confirmed in Australia (Northern Territory and Queensland), China (Hainan, Hunan, Guangdong, and Guangxi), Indonesia (Bali, Halmahera, Kalimantan, Java, Papua Province, Sulawesi, and Sumatra), Jordan, Lebanon, Malaysia (Peninsular and Sarawak), Mozambique, Oman, Pakistan, the Philippines (Mindanao), and Taiwan (Butler, 2013;Garcia et al. 2013;Molina et al., 2010;Ordonez et al., 2015;Ploetz, 2006;Ploetz et al., 2015). Control strategies of this pathogen are based on visual monitoring for early symptom appearance, eradication of infected plants and isolation of infested areas to reduce pathogen dissemination. However, these strategies are often impractical and therefore does not carried out (Dita et al., 2010).
Based on Vegetative Compatibilty Group (VCG), Foc have been separated into 24 vegetative compatibility groups (VCGs) (Ghag et al., 2015). Finally, various identification tools have been used to separate Foc into a number of clonal lineages to their grouping based on VCGs (Bentley et al., 1998). Till now, Foc separated into two clades and eight lineages with the majority of groups present in Asia (Ghag et al., 2015). Phenotypic and genotypic analyses of worldwide collections of Foc suggest that Southeast Asia is the centre of origin of Foc (Fourie et al., 2009) and the pathogen has been introduced into new regions from there (Stover, 1962). The tropic race 4 strain of Foc belongs to a single group of VCG 01213/16 complex, whereas the subtropic race 4 isolates belong to VCG 0120, 0121, 0122, 0129 and 01211 (Buddenhagen, 2009).
The development of a rapid and reliable PCR diagnostic for Foc TR4 (VCG 01213) using Random Amplification of Polymorphic DNA (RAPD), Restriction Fragment Length Polymorphisms (RFLPs), Sequence Characterized Amplified Region (SCAR), Elongation Factor (EF1α) gene, Single Nucleotide Polymorphisms (SNPs) based on the Intergenic spacer (IGS) and small secreted protein (Six, Secret in xylem) have been developed for the identification of Foc using diagnostic traits that are directly linked to pathogenicity or virulence would provide another powerful approach for identification of soil-borne pathogenic fungi such as Foc TR4 (Lin et al., 2008;Liao et al., 2009;Dita et al., 2010;Lin et al., 2012;Li et al., 2013;Guo et al., 2014). Therefore, the main objective of this study is to evaluate the specific primer sets development as well as its specificity for Foc TR4 (VCG 01213) detection originating from different banana production areas in Indonesia.

Isolates collection
In total, 50 monosporic cultures of Foc isolates originating from different banana production areas in Indonesia (Bangka, West Java, Central Java, East Java, Kalimantan, and Sumatera) are analysed.
The cultivars, genotypes and VCG of all Foc isolates are presented in Table 2. VCG tests are performed for all of the Foc isolates from different banana production areas have been done by Wibowo et al. (2011), including VCG 01213 which are belong to Foc TR4 (Table 2).

DNA Extraction
To obtain the mycelial mass, the isolates are grown on Potato Dextrose Broth medium (PDB) in 100 ml Erlenmeyer flasks for 7 days at 25ºC. The isolates are shaken continuously for a period depending on each isolate. After 7 days, the mycelial mass of each isolate is filtered for the extraction of DNA. Filter paper is used to collect the mycelia and they are grinded in CTAB buffer using a mortar and pestle. The extraction of DNA from the isolates is performed using the CTAB method with some modification. PCR product is visualized on a 1% agarose gel stained with ethidium bromide viewed under UV light.

Identification Foc Isolates Using FocEf3 Primer Set
Firstly, to make sure that DNA samples of fifty isolates are Foc, FocEf3 primer set that is designed by Widinugraheni et al. (2015) used for Foc detection yield an amplification product of 600 bp using the following programme: 94ºC for 5 min followed by 30 cycles consisting of 94ºC for 30 sec, 57ºC for 30 sec, and 72ºC for 2 min 30 sec followed by an additional extension time at 72°C for 5 min.

Evaluation Test of Foc Isolates Using Specific Primer Design of Foc Race 4 and Foc TR4
All isolates are tested using specific primer Foc-1/ Foc-2 (Lin et al., 2008) to detect Foc race 4. This primer yield an amplification product of 242 bp using the following programme: 95ºC for 5 min followed by 33 cycles consisting of 95ºC for 1 min, 55ºC for 1 min, and 72ºC for 3 min followed by an additional extension time at 72ºC for 10 min. The amplified isolates using Foc-1/Foc-2, are tested using specific primer sets to detect Foc TR4 i.e. TR4-F/ TR4-R (Dita et al., 2010), Six-1c (Widinugraheni et al., 2015) and TR4-F2/TR4-R1 (Bentley et al., 2003). TR4-F/TR4-R yield an amplification product of 463 bp with the following programme: 95ºC for 5 min followed by 30 cycles consisting of 95ºC for 1 min, 60°C for 1 min, and 72ºC for 3 min followed by an additional extension time at 72ºC for 10 min. Six-1c yield an amplification product of 884 bp with the following programme: 94ºC for 5 min followed by 30 cycles consisting of 94ºC for 30 s, 57ºC for 30 s, and 72ºC for 2 min 30 s followed by an additional extension time at 72ºC for 5 min. TR4-F2/TR4-R1 primer set that also used in this study according the combination and modification of methods developed by Bentley et al. (2003), yield an amplification product of 1400 bp with the following programme: 95ºC for 2 min followed by 30 cycles consisting of 95ºC for 30 sec, 68ºC for 1 min 30 sec, and 72ºC for 3 min followed by an additional extension time at 72ºC for 10 min.

Identification Foc Isolates Using FocEF3 Primer Set
In total, 48 isolates are confirmed as Foc isolates based on the test using FocEf3 primer set which is designed by Widinugraheni et al. (2015) for Foc except Cjr-2 and Lmp-4 isolates that probably contaminant (Table 1).

Evaluation Test of Foc Isolates Using Specific Primer Design of Foc Race 4 and Foc Tropic Race 4
Lin et al. (2008) designed a specific primer set which is derived from RAPD fragment specific to Foc race 4 isolates from Taiwan are amplified by the random primer OPA02404 and designed a specific primer set called Foc-1/Foc-2 (Foc242) from the OPA02404 nucleotide sequences is used in this study. The Foc-1/Foc-2 (Foc242) had been confirmed with high specificity to detect Foc race 4 (Foc race 4) (Lin et al., 2008). From 50 Foc isolates, Foc-1/Foc-2 could amplify 34 isolates with a percentage of 68%, but not to the other 16 isolates (Table 1).
TR4-F/TR4-R primer set derived from sequences of IGS region to study genetic diversity of Foc, with the aim of identifying Single Nucleotide Polymorphisms (SNPs) for specific primer design for Foc TR4 detection. However, the results showed that the higher SNP frequency of the IGS region provides a rich source of genetic diversity in Foc (Dita et al., 2010). From 50 Foc isolates have been tested by TR4-F/TR4-R, 35 Foc isolates could be amplified by TR4-F/TR4-R with a percentage of 70%, but not to the other 15 isolates (Table 1).
Six-1c specific primer set that is designed by Widinugraheni et al. (2015) with some selection of several effectors on Foc TR4 (II5) strain genome based on the homology of Sixgeneis used in this study also capable in detecting Foc TR4. From the result, Six-1c could amplify 25 Foc isolates with a percentage of 50%, but not to the other 25 isolates (Table 1).
Fungi, like all living organisms, have the ability to adapt in response to changing or new environments. Environmental changes exert selection pressure on an organism (McDonald, 1997), and only individuals that adjust to change are able to succeed. The capacity   of population of pathogens to adapt is determined, in part, by their diversity. As the gene pool that a population can sample increases, so does its adaptability to a changing environment or a new host genotype (McDonald & McDermott, 1993).
The evolutionary history of Foc is very complex. Based on the phylogenetic studies of Foc divided taxon into several lineage representing two of four F. oxysporum clade, indicating that the polyphyletic of Foc more closely related to other F. oxysporum forma specialist than Foc lineage. This led to a speculation that the formation of a new pathogen from F. oxysporum maybe come from a member of the pathogens and non-pathogens from this species (Fourie et al., 2011). Fourie et al. (2011) also confirm that co-evolution between plant and horizontal gene transfer is the evolutionary history of Foc and showed that there is a lot of diversity on Foc strain that should be studied and need further research and surveys against the evolution of this pathogen.
Genetically, Foc have ability to infect specific banana cultivar, so that it can be grouped into biological races (Waite & Stover, 1960;Su et al., 1986). Based on the tested Foc isolates from Indonesia and some previous research in grouping Foc based on the PCR-based methods using specific primer sets, it turns out that there is not enough to grouping different strains of Foc based on the races. Foc is classified into two clades A and B based on the evolutionary origins. Clade A group of Foc, co-evolved with bananas having A genome, whereas clade B evolved along with their host banana plants having purely B genome or mixed A and B genomes. Currently, the banana disease causing strains of Foc are evolved from both clade A and clade B lineages (Fourie et al., 2009). In order to characterize these genetically related populations, they have now been grouped into same VCGs based on the formation of a heterokaryon (Puhalla, 1985).
The VCG gives a fair idea of the genetic diversity and evolution of the pathogen. However, the use of VCG as a means to classify Foc is also considered incomplete as one race since it can comprise of more than one VCG (e.g. VCG 0124 and VCG 0125 belong to race 1) or one VCG may occur in multiple races (e.g. VCG 0124 occurs in both race 1 and race 2). In addition, few of the VCG groups are cross compatible, giving rise to VCG complexes making it more difficult for pathotype identification. Till now, 24 VCGs of Foc have been identified with further separation into two clades and eight lineages originated from Asia (Ghag et al., 2015).
This result suggested that there are a large amount of diversity seen in Foc strains have to be discovered and that additional surveys and research are needed for the full appreciation of the evolution of this pathogen. Therefore, in order to pinpoint potential species boundaries to elucidate the true relationships among the VCGs and lineages of F. oxysporum f. sp. cubense, then, the diversity of F. oxysporum complex needs to be fully characterized (Fourie et al., 2009).

CONCLUSION
It showed that tropic and subtropic race 4 of Foc have been widely distributed in some regions in Indonesia. Based on the tested Foc TR4 isolates (VCG 01213) as control, TR4-F/TR4-R have the highest specificity to Foc TR4 isolate detection. Meanwhile, TR4-F2/TR4-R1 has the lowest specificity to Foc TR4 isolate detection. Further research should develop a specific primer set for detecting Foc TR4 and the most damaging strain of Foc based on genetic diversity. Therefore, in studying Foc especially Foc TR4 strain should focus on the specificity of genetic based on molecular data.