Race and Virulence Determination of Fusarium Oxysporum F. SP. Cubense Isolates From Sidomulyo Village of Bantul, YOGYAKARTA

Banana is one of the important fruit crop in Village of Sidomulyo, Bantul, Yogyakarta. One of important diseases which become the constraint in development of banana is Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc). This fungus has high race diversity and virulence, so that it required early detection for prevention and control of disease. This experiment was aimed to figure out race and virulence of Foc isolates from Village of Sidomulyo, Bantul, Yogyakarta. The 13 tested isolates were isolates of PR11, PKJ20, RU20, PR30, AH40, PKJ40, A41, RB42, PR43, RU51, A60, RP60, and A80. Race was molecularly detected using two types of primers, i.e. General Foc primer FocEf3 and specific primer for race 4 (Foc-1/Foc-2). Virulence test was performed on banana seedlings of Ambon Kuning cultivar using Completely Randomized Design (CRD) with 14 treatments and 4 repetitons. The observed parameters were external and internal symptoms, calculation of disease severity index and disease intensity. Data were analyzed using variance and further test of Duncan Multiple Range Test (DMRT) at 5 % level. The results showed that all isolates were Foc and 9 of 13 isolates were grouped into race 4, i.e. A80, RP60, PR11, A41, AH40, PKJ40, PR30, RB42, and PR43. The highest and lowest virulences were consecutively expressed by PR30, RB42, RU51, RP60, PR43, PKJ40, PR11, A41, AH40, RU20, PKJ20, A60, and A80, with severity index on leaves and rhizomes ranging 1.61-2.91 and 2.25-7, respectively. IntisariPisang merupakan tanaman buah unggulan di Desa Sidomulyo Kecamatan Bambanglipuro, Kabupaten Bantul. Salah satu penyakit penting yang menjadi kendala dalam pengembangan pisang adalah layu fusarium yang disebabkan oleh jamur Fusarium oxysporum f. sp. cubense (Foc). Jamur ini memiliki keragaman ras dan virulensi yang tinggi, sehingga deteksi dini diperlukan untuk pencegahan dan pengendalian penyakit. Penelitian ini bertujuan untuk mengetahui ras dan virulensi isolat Foc asal Desa Sidomulyo, Kecamatan Bambanglipuro, Kabupaten Bantul. Isolat yang diuji sebanyak 13 isolat, yakni isolat PR11, PKJ20, RU20, PR30, AH40, PKJ40, A41, RB42, PR43, RU51, A60, RP60, dan A80. Pengujian ras secara molekuler dengan menggunakan dua jenis primer yakni primer Foc in general FocEf3 dan primer spesifik ras 4 Foc-1/Foc-2. Uji virulensi pada bibit kultivar ambon kuning dengan menggunakan Racangan Acak Lengkap (RAL) yang terdiri dari 14 perlakuan dan 4 ulangan. Parameter yang diamati berupa pengamatan gejala luar dan gejala dalam, penghitungan indeks keparahan penyakit dan intensitas penyakit. Analisis data menggunakan sidik ragam dan uji lanjut Duncan Multiple Range Test (DMRT) pada taraf 5 %. Hasil pengujian menunjukkan bahwa semua isolat merupakan isolat Foc dan dari 13 isolat yang digunakan terdapat 9 isolat yang merupakan ras 4 yakni isolat A80, RP60, PR11, A41, AH40, PKJ40, PR30, RB42, dan PR43. Isolat yang memiliki virulensi tertinggi sampai terendah berturut-turut adalah PR30, RB42, RU51, RP60, PR43, PKJ40, PR11, A41, AH40, RU20, PKJ20, A60, dan A80, dengan indeks keparahan pada daun berkisar 1,61−2,91 dan indeks keparahan pada bonggol 2,25−7.


INTRODUCTION
Banana is one of important economic horticultural crops which was cultivated by farmers in Village of Sidomulyo, Bantul, Yogyakarta. Data of fruit crops in this village placed it as the most planted one, i.e. about 15,957 plants, followed by 2,706 plants of mango, 2,288 plants of melinjo and 1,754 plants of rambutan (BKP3, 2016). According to data of BPS (2016), banana production in Village of Sidomulyo was equivalent to total of productions in two other villages within District of Bambanglipuro, namely 200.9 ton in Sidomulyo, 100.5 ton in Mulyodadi and 100.4 ton in Sumbermulyo.
The development of banana plantation can not be separated from the prevalence of crop disease. One of important disease invading banana crop is Fusarium wilt caused by Fusarium oxysporum Schlecht f. sp. cubense (Foc) (E.F. Smith) Snyder & Hansen (foc) (Ploetz, 2006;Ghag et al., 2015). Diseased plant will express the symptoms of yellowing leaves from the lower ones, wilt and longitudinal section of rhizomes showed the brown blackish stripe towards up all parts of plant through pseudostem (Semangun, 2000). This disease is very dangerous and threatening banana industry in the world (Moore et al., 1995;Visser, 2010). It has been reported destroying more than 40,000 ha of banana orchards in Central America an South America for period of 50 years (Su et al., 1986). In Indonesia, the infection has been detected in whole regions from Aceh to Papua Jumjunidang et al., 2012) and abolish thousands hectares of commercial and private banana plantations (Nasir et al., 2005).
The high diversity in race and virulence of Foc enabled early detection is required very much in efforts of prevention and controlling of Fusarium wilt disease. Molecular identification from various isolates can be referred as basic of race categorization on Foc (Kuswinanti et al., 2011). This research was aimed to figure out race and virulence of Fusarium oxysporum f. sp. cubense (Foc) isolates from Village of Sidomulyo, Bantul, Yogyakarta.

Molecular Identification on Race of Fusarium oxysporum f. sp. cubense Isolates
Sample collection. Samples were randomly collected from 7 subvillage in Village of Sidomulyo, District of Bambanglipuro, Regency of Bantul, namely Ponggok, Pinggir, Plebengan, Plemantung, Sirat, Selo, and Cangkring. One infected cultivar was considered as one sample, whereas if more diseased cultivars were found, the samples were taken from each cultivar.
Sampled banana plants showing fusarium wilt symptoms were cut by chopping the pseudo-stem about 20−30 cm from neck of rhizome with approximately 5×15 cm in size. The internal part of infected pseudo-stem expressed reddish or brownish color. Afterwards, the vessel spindles were gently pull out to separate them from tisses, air-dried, put on filter paper, covered with steril tissue paper, kept into envelopes and then put into closed plastic box containing silica gel. The number of obtained samples were 13 as shown in Table 1.
Isolation and purification of Fusarium oxysporum f. sp. cubense isolates. The air-dried tissues of pseudostem were cut about 0.5-1 cm, cultured on Potato Dextrose Agar (PDA) medium containing 1 drop of 25% lactic acid and then incubated under room temperature for 7 days (25ºC-27ºC). The isolated fungi were then purified using single spore isolation technique. This technique was performed by diluting the fungal colony with 10 ml of sterile aquadest and vortexing for 2 min. Furthermore, fungal suspension was streaked onto water agar (WA) medium using ose needle, incubated for 15 h at room temperature (25ºC-27ºC) , and then the single germinating spore was transferred onto PDA medium and incubated for 7 days at room temperature (25ºC-27ºC). Vol. 22 No. 1 74 DNA isolation from isolates of Fusarium oxysporum f. sp. cubense. Fungi were cultured in 40 ml of Potato Dextrose Broth (PDB) medium in Erlenmeyer and stirred using shaker for 1 week at room temperature. DNA was extracted using CTAB method of Subandiyah (2003).

DNA amplification.
DNA amplification was carried out using two primers, i.e. primer of Foc in general FocEf3 (Widinugraheni, 2015) to ensure that all tested isolates were Fusarium oxysporum f. sp. cubense and specific primers for race 4 of Foc-1/Foc-2 (Lin et al., 2009). PCR program for those primers were shown in Table 2 and Table 3, respectively.

Detection of Fragments by Electrophoresis
The next stage was electrophoresis on PCR product using 1 % of agarose gel in TBE 1x solution. As much of 5 µl PCR product was pipetted into well of agarose gel as well as 5 µl of 100 bp marker to indicate the size of DNA bands. Electrophoresis was run for 45 min at 50 volt. Gel was stained by dipping in ethidium bromide (EtBr) solution for 15 min and then rinsed with sterile aquadest. DNA bands were visualized and documented on UV transluminator.

Preparation of Inoculum
Fusarium oxysporum f. sp. cubense isolates were culture on slant agar in reaction tubes containing PDA medium and incubated for 7 days. Conidia suspension was prepared by adding 10 ml of sterile aquadest into test tubes and harvesting the mycelia using ose needle. Afterwards, conidia concentration was adjusted to 10 7 conidia/ml water.  Table 3. PCR program using specific primer for race 4 of Foc-1/Foc-2 (Lin et al., 2008)

Inoculation of Foc on Banana Seedling of Ambon Kuning
This experiment was completely randomized design (CRD) with 14 treatments and 4 repetitions. The used banana plants were 6-month tissue culture seedlings of Ambon Kuning cultivar obtained from Banana Germplasm Orchard of Yogyakarta. The seedlings were planted in polybag containing sterile soil. Artificial inoculation was performed by injuring the rooting and rhizome areas using sterile scalpel and then pouring the conidia suspension into those areas. Control used sterile water. The inoculated seedlings were kept in the glass house.

Virulence Assay
Observation on external symptom. The observation was weekly conducted for 7 weeks. The observed parameter was wilt symptom on leaf, and then analyzed according to leaf symptom index (LSI) using method of Mak et al. (2004) which had been modified by Kiswanti et al. (2010) as displayed in Table 4.
Observation on internal symptoms. The observation of rotting symptom on rhizome (as known as Rhizome Discoloration Index/RDI) was carried out at 7 th week after inoculation, and then analyzed according to method of Mak et al. (2004) which had been modified by Kiswanti et al. (2010) as shown in Table 5.

Calculation of Disease Severity Index
Disease Severity Index (DSI) was overall counted based on RDI and LSI data using following formulation: Virulence of isolates was determined by results of LSI and RDI following method of Mak et al. (2004) which has been modified by Kiswanti et al. (2010) as shown in Table 6.

Observation of Disease Development
Disease development was observed according to number of yellowing leaves on one plant (Wibowo et al., 2001). Scoring for observation of yellowing leaves on banana plants was based on the following Table 7 . Having leaf observation, the disease intensity was calculated with formulation as below: Note: IP : Disease Intensity ni : Number of leaves on each corresponding score vi : Score of disease on corresponding leaf Z : Highest score N : Number of observed leaves

Data Analysis
Disease intensity from first week until the 7 th week were analyzed using ANOVA. If there is significantly different, it was further analysis using Duncan Multiple Range Test (DMRT) at 5 % level. No discoloration/rotting on rooting area, discoloration found on root branches only 2 Discoloration/rotting up to 5% on rhizome 3 Discoloration/rotting up to 6-20% on rhizome 4 Discoloration/rotting up to 21-50% on rhizome 5 Discoloration/rotting up to >50% on rhizome 6 Rotting on whole parts of rhizome and rooting area 7 Plant died

Molecular Identification on Race of Foc
The result showed that all isolates could be amplified with primers of Foc in general FocEf3 which was indicated by the presence of DNA bands at 600 bp in size (Figure 1a). It could be concluded that all tested isolates were Fusarium oxysporum f. sp. cubense.
The electrophoresis of PCR product using specific primers for race 4 of Foc-1/Foc-2 expressed that 9 of 13 isolates could be amplified at 242 bp in size (Figure 1b). Primer of Foc-1/Foc-2 had high specificity in detecting the isolates of Foc race 4 (Foc subtropical race 4 (SR4) and tropical race 4 (TR4) (Lin et al., 2009). However, isolates of PKJ20, A60, RU20 and RU51 were not amplified using those primers. Those isolates were assumed to be categorized into Foc race 1 since the infected hosts were cultivars of pisang Koja/pisang susu (AAA), pisang ambon kuning (AAA) and pisang raja uter (AAB). Jeger et al. (1995) reported that Foc race 1 was pathogenic against banana cultivar having genomes of AAA and AAB.

Virulence Assay on Isolates of Foc on Banana Seedling of Ambon Kuning
Disease severity could be considered as one of foundation in determining the virulence level of pathogen. It was measured as percentage of infected part of plants such as roots, leaves or stems with corresponding symptoms generated by given pathogen (Pariaud et al., 2009). The level of disease severity using isolates of Foc on banana seedlings in this experiment could be seen by observating external symptoms (yellowing or wilting leaves) and internal symptoms (rotting of rhizome). This research documented LSI in range of 0.18-3.13 and RDI of 0-7 in range as expressed in Table 8.
Analysis of DSI against using LSI and RDI data showed that 8 of 13 isolates were very virulent, i.e. isolates of PR11, A41, AH40, PKJ40, RU51, PR30, RB42, and PR43; while other isolates (PKJ20, A80, RP60, A60, and RU20) were virulent. The difference in virulence occurred because of the difference in biological, chemical, genetic and ability of asexual spore reproduction on each isolate (Groenewald, 2005;Jumjunidang et al., 2011). All of high virulent isolates were grouped into race 4 excluding isolate of RU51. Su et al. (1986) and Ploetz (2006) explained that Foc race 4 was the most terrible and virulent race since it was able to infect all types of banana including those which were susceptible against race 1 and 2.
The difference in categorization of disease severity level could also be viewed from LSI and RDI data on isolate of RU20 (Table 8). LSI data of this isolate indicated that it belonged to high virulent category, while RDI data revealed that isolate of RU20 was virulent. However, the categorization of virulence referred to RDI data since the infection of pathogen initiated from rooting and rhizome areas. According to Ploetz (2000), Foc penetrated to plant tissues through root and then developed towards stem and extend to vessel tissue. Fungal development in tissue affected the interruption of water and nutrient flow from soil so that the plants was getting wilt which was indicated by the yellowing of bottom leaves. The control plant expressed LSI data about 0.18, which was obtained from the number of yellowing leaves during observation. However, these yellowing leaves were assumed due to the aging stage of leaves. It was proved by RDI data which did not find the rotting symptoms either on rhizome or rooting areas.

Disease Progress
The results showed that the symptom of Fusarium wilt disease on banana seedling of ambon kuning emerged on 1 st week after inoculation, excluding for  Table 7. Scoring grade of yellowing leaves for observation of disease intensity control ( Figure 2). This early emergence of symptom might be caused by pre-injuring on plant rooting and high concentration of applied inoculum. According to Agrios (2005), the wounding of plant would enable the pathogen to penetrate and introduce into plant tissue. During infection, pathogen would grow, reproduce and colonize the plant. The successful infection process would generate the symptom on plant. The used inoculum density in this experiment could be considered as high concentration, i.e. 10 7 conidium/ml water. Mak et al. (2004) reported that conidium density of Foc with concentration of 5×10 6 conidium/ml water had been able to appear the symptom at 10 th day after inoculation. Therefore, the chance and ability of pathogen to infect with the higher concentration (10 7 conidium/ml water) were increasing so that the emergence of symptom would be earlier. It was paralel to Agrios (2005) and Riska et al. (2012) who explained that high inoculum density would fasten disease development, improve the ability to generate symptom, and increase disease severity.
It was found the difference in development of disease intensity corresponding to each isolate of Foc until 7 th week observation. Disease intensity started to quickly increase at 3 rd week after inoculation. At the last week after inoculation, it could reach about 58.71% to 97.73%. The plant which was inoculated with isolate of PR30 expressed the disease intensity about 26.01% at 3 rd week and up to 97.73% at 7 th week. Meanwhile, disease intensities caused by isolate of A80 were about 8.8% and 58.71% at 3rd and 7 th week, respectively.   Means of disease intensity at 7 th week after inoculation showed that the highest disease intensity was found on treatment with isolate of PR30 about 97.73%, followed by isolates of RB42, RU51, RP60, PR43, PKJ40, PR11, A41, AH40, RU20, PKJ20, A60, A80, and control about 90.87%; 87.50%; 85.49%; 84.98%; 82.52%; 82.35%; 81.59%;79.79%; 74.46%; 68.75%; 64.94%; 58.71%; and 0%, respectively. It could be concluded that isolate of PR30 had highest virulence level and isolate of A80 was the lowest (Table 9).
The variance analysis in Table 9 indicated that treatment with PR30 was not significantly different to treatments with isolates of RB42, RU51, RP60, PR43, PKJ40, PR11, A41, AH40, and RU20, but it was significantly different to treatments with isolates of PKJ20, A60 and A80.
The development of disease intensity was supposed to be related to factors of pathoen and plant. Pathogen in this case was correlated to high density of inoculated inoculum, so that it had high capability to cause the disease. According to Purwanti et al. (2008), high density of Foc conidia was effective in raising disease intensity based on the symptom of yellowing and wilting leaves on Abaca plant. In addition, the virulence of pathogen also influenced disease intensity. The highest disease intensity was found on isolate of PR30 which was race 4 of Foc. It was recognized having high virulence in attacking all banana cultivars. High disease intensity was also presented by isolate of RU51 which was considered as race 1 of Foc. Previously, Bentley et al. (1998) explained that race 1 of Foc could affect Gros Michel cultivar and even destroy banana industry in the world. Recently, Hermanto et al. (2013) reported that pisang ambon kuning belonged to Gros Michel group which might be infected with high percentage as well. The ability in producing toxin of fusaric acid also affected the virulence of each tested Foc isolate. The higher production of fusaric acid, the higher virulence and disease intensity of isolates. It was indicated by the development of wilting or yellowing leaves (Dong et al., 2012). Fusaric acid was a phytotoxin produced by Foc which disrupted the permeability of plant membrane, inhibited the oxygen taking and had important role as causal agent of yellowing leaves and enhanced the aging process on infected plants (Dong et al., 2014).
Susceptible plant and less nutrient soil could improve the emergence of disease when they were inoculated with virulent pathogen. This experiment used cultivar of ambon kuning which was one of susceptible cultivars against Foc and the plants were not treated with additional nutrients so that they did not sturdy grow and were getting susceptible against Foc.

CONCLUSION
This investigation concluded that all tested isolates were Foc and 9 of 13 isolates were categorized into race 4, i.e. A80, RP60, PR11, A41, AH40, PKJ40, PR30, RB42, and PR43. All isolates could be successively listed from the most to the least virulent, i.e. PR30, RB42, RU51, RP60, PR43, PKJ40, PR11, A41, AH40, RU20, PKJ20, A60, and A80, with LSI ranged 1.61−2.91 and RSI was about 2.25−7. High virulence and abundance of race 4 from obtained isolates of Sidomulyo Village are expected to increase the early awareness of farmers in cultivating banana plant. The use of healthy and non-susceptible banana seedlings in Foc-infected soils was suggested to farmers in Village of Sidomulyo, District of Bambanglipuro, Regency of Bantul.

ACKNOWLEDGEMENT
Author would like to thank to The Royal Netherlands Academy of Arts and Sciences Scientific Programme Indonesia-Netherlands (KNAW SPIN) -The Indonesian Banana and Agricultural Institute for Extension and Human Resource Development, Ministry of Agriculture, for funding support of this research. This manuscript is part of the thesis entitled "Race and Virulence Determination of Fusarium oxysporum f. sp. cubense Isolates from Sidomulyo Village of Bantul, Yogyakarta."