4. 649 Geminiviruses (rika).cdr Geminiviruses Associated with the Weed Species , and from Java, Indonesia Ageratum conyzoides Centipeda minima, Porophyllum ruderale, Spilanthes iabadicensis RIKA MELIANSYAH , SRI HENDRASTUTI HIDAYAT *, KIKIN HAMZAH MUTAQIN1,2 1 1AND 1 2 Department of Plant Protection, Faculty of Agriculture, Institut Pertanian Bogor, Darmaga Campus, Bogor 16680, Indonesia; Department of Agrotechnology, Faculty of Agriculture, Universitas Pajajaran, Jalan Raya Bandung - Sumedang Km 2140600, Jatinangor, Bandung UBR 40600, Indonesia Geminivirus Geminivirus Geminivirus Geminivirus Ageratum conyzoides Centipeda minima A. boehmerioides Porophyllum ruderale Spilanthes iabadicensis Geminivirus Geminivirus Begomovirus Geminivirus Geminivirus Geminivirus Geminivirus Geminivirus Ageratum conyzoides , Centipeda minima A. boehmerioides Porophyllum ruderale Spilanthes iabadicensis Geminivirus Geminivirus GenBank Geminivirus Geminivirus Begomovirus Geminivirus has a wide host range including cultivated plants and weeds. Infected weeds may play an important role in disease epidemic. Unfortunately, little is known about weeds species that may serve as alternative host for . This research was conducted to identify on weeds around chili pepper field to study their potential role as virus reservoir. Field surveys were conducted to chilli pepper growing area in West and Central Java Provinces, and The Special Province of Yogyakarta during 2009 to collect symptomatic weed plants. infection was detected using PCR technique from 9 weed samples, i.e. 5 samples from Bogor (AgrBgr), Sukabumi (AgrSkm), Magelang (AgrMgl), Sleman (AgrJgy), and Garut (AgrGrt); from Magelang (CtpMgl); from Sleman (AcpJgy); from Bogor (PrlBgr); from Magelang (SplMgl). Further genetic analysis showed that those geminiviruses can be differentiated into 2 clusters, showing the possible genetic differences among them. They neither have a close relationship with other geminiviruses published earlier in the GenBank, indicating weed infecting collected in this study is possibly a distinct . Key words: , , polymerase chain reaction, weed species memiliki kisaran inang yang luas termasuk berbagai tanaman budi daya maupun gulma. Hingga saat ini pengetahuan tentang jenis-jenis gulma yang berpotensi menjadi sumber masih sangat terbatas. Penelitian ini dilakukan dengan tujuan mengidentifikasi pada gulma-gulma yang tumbuh di sekitar pertanaman cabai dalam upaya mempelajari perannya sebagai sumber inokulum virus. Survei lapangan telah dilakukan pada tahun 2009 ke beberapa daerah penanaman cabai di Provinsi Jawa Barat, Jawa Tengah, dan Daerah Istimewa Yogyakarta untuk mengumpulkan gulma-gulma yang menunjukkan gejala. Infeksi berhasil dideteksi menggunakan teknik PCR dari 9 sampel gulma, yaitu 5 sampel masing-masing dari Bogor (AgrBgr), Sukabumi (AgrSkm), Magelang (AgrMgl), Sleman (AgrJgy), dan Garut (AgrGrt) dari Magelang (CtpMgl), dari Sleman (AcpJgy), dari Bogor (PrlBgr), dari Magelang (SplMgl). Analisis genetika lebih lanjut menunjukkan bahwa yang menginfeksi gulma-gulma tersebut dapat dibedakan menjadi 2 kelompok besar yang merupakan indikasi adanya keragaman genetika di antara mereka. yang menginfeksi spesies gulma yang berbeda tersebut tidak memiliki hubungan kekerabatan yang dekat dengan geminivirus lain yang dilaporkan dalam sehingga yang diidentifikasi dari spesies gulma tersebut merupakan yang berbeda.. Kata kunci: , , polymerase chain reaction, gulma Geminiviruses are single-stranded DNA viruses with geminate particle morphology. They are classified into four genera ( , and ) on the basis of host range, insect vector and genome organization (Fauquet and Stanley 2005). Most of the geminiviruses are transmitted by whiteflies and belong to the genus . These species have been reported to cause significant economic yield losses to many crops in tropical and subtropical regions of the world. Member of was known to have enormous diversity resulting from their widespread geographic distribution and host adaptation (Varma and Malathi 2003). Diseases caused by in Indonesia has been reported including those infecting tobacco Mastrevirus, Curtovirus, Topocuvirus Begomovirus Begomovirus Begomovirus Begomovirus (Trisusilowati . 1990), tomato (Sukamto . 2005; Kon . 2006; Santoso . 2008), chilli pepper (Sulandari . 2006; Hidayat . 2006; Trisno . 2009), and also a weed species (Haerani and Hidayat 2003; Kon . 2007). Among those diseases, the most concerned one is pepper yellow leaf curl disease which induces symptoms involving foliar chlorosis and curling, reduced leaf size, inhibited fruit set and abnormal fruit. Emergence of pepper yellow leaf curl disease in Indonesia was first reported in 1999 in West Java (Rusli . 1999), and in 2003 the disease had been widely spread in Java with the highest incidence and severity occurred particularly in Central Java (Sulandari . 2006). Census data for the period from 2001 - 2003 shows that the disease has undergone a 4.6 fold increase between 2001 and 2002 and a 2.5 fold increase between 2002 and 2003 (Indonesian Ministry of et al et al et al et al et al et al et al Ageratum conyzoides et al et al et al *Corresponding author, Phone: +62-251-8629363, Fax: +62-251-8629362, E-mail: srihendrastutihidayat@gmail.com ISSN 1978-3477, eISSN 2087-8575 Vol 5, No 3, September 2011, p 120-124 I N D O N E S I A Available online at: http://www.permi.or.id/journal/index.php/mionline DOI: 10.5454/mi.5.3.4 Agriculture 2003, unpublished data). It was believed that two main factors may contribute to the spread and distribution of the disease in Indonesia, i.e. fluctuation of whitefly population and the presence of alternative plants that may act as virus reservoirs. It was reported previously that weed species may serve as alternative hosts for geminiviruses. Yang . (2008) identified a distinct species from from Fujian province, China; whereas Wu . (2007) was successfully isolated a monopartite geminivirus from Three weed species ( spp., , and ) w e r e i d e n t i f i e d a s p o t e n t i a l 's alternative host in Jamaica (Roye and McLaughlin 1997); meanwhile in Indonesia Sulandari . (2006) reported that , , , were very susceptible to geminivirus infection. Thus, to build more information on the potency of weed species as virus reservoirs especially for pepper yellow leaf curl disease in Java, surveys were carried out in 2009 in West and Central Java. To this aim, weed samples showing leaf curl and yellow vein symptoms were collected from different chilli pepper growing area. In this paper, we report the identification of geminivirus associated with yellow disease of 4 weed et al Begomovirus Emilia sonchifolia et al M. coromandelianum from Guangdong, China. Sida Macroptilium lathyroides Wissadula a m p l i s s i m a Begomovirus et al Hyptis brevipes Physalis floridana Crotalaria juncea Ageratum conyzoides species: , and . Naturally infected weed species with yellow vein symptoms were observed in West Java (Bogor, Sukabumi, Garut), Central Java (Magelang), and Yogyakarta (Sleman). The specific symptoms was observed in weed species ( , ), s p e c i e s ( ) , species ( ), and species ( ) Viral DNA was extracted from all samples as described by Kon . (2002). Total DNA was extracted from symptomatic leaves according to Kon (2002). The DNA pellet was resuspended in 100 µL TE buffer. Amplification of geminivirus genome was proceeded using a pair of specific primers designed for the amplification of coat protein gene, CP Protein-V1 (5' TAATTCTAGATGTCGAAGCGACCCGCCGA 3') and CP Protein-C1(5' GGCCGAATTTCTTAATTTT GAACAGAATCA 3'). These specific primers were A. conyzoides Centipeda minima, Porophyllum ruderale, Spilanthes iabadicensis Compositae A. conyzoides, C. minima, E. prostrate, P. ruderale S. iabadicensis, G. peruviana E u p h o r b i a c e a e C r o t o n h i r t u s Convolvulaceae Ipomoea triloba Onagraceae Ludwigia peruviana . et al et al. MATERIALS AND METHODS Virus Sources and DNA Extraction. PCR-Based Detection Using Geminivirus Specific Primers. Volume 5, 2011 Microbiol Indones 121 Geminivirus Acronim Geographic Location Sequence Length (bp) GenBank Accession No. Ageratum conyzoides -Bogor AgrBgr Indonesia : Bogor, West Java 864 NS Ageratum conyzoides -Sukabumi AgrSkm Indonesia : Sukabumi, West Java 993 NS Ageratum conyzoides -Magelang AgrMgl Indonesia : Magelang, Central Java 868 NS Ageratum conyzoides -Sleman AgrJgy Indonesia : Sleman, Jogyakarta 843 NS Spilanthes iabadicencis- Magelang SplMgl Indonesia : Magelang, Central Java 890 NS Centipeda minima- Magelang CtpMgl Indonesia : Magelang, Central Java 756 NS Porophyllum ruderale -Bogor PrlBgr Indonesia : Bogor, West Java 832 NS Bean yellow dwarf virus BYDV South Africa 2566 DQ458791 Tomato leaf curl Java virus TLCJV Indonesia : Java 2752 AB100304 Tomato leaf curl Java virus-[Ageratum] TLCJV- [Ageratum] Indonesia : Java 2747 AB162141 Tomato leaf curl Malaysia virus TLCV Malaysia 2754 AF327436 Tomato leaf curl Laos virus TLCV Laos 2748 AF195782 Ageratum yellow vein Taiwan virus AYVV Taiwan 2734 AF307861 Ageratum yellow vein China virus-[Hn2] AYVV China 2768 AJ495813 Chilli leaf curl virus-[Multan] ChilCVA Pakistan 2754 AF336806 Pepper leaf curl Bangladesh virus PepLCV Bangladesh 2753 AF314531 Pepper yellow leaf curl Indonesia virus PepYLCV Indonesia : West Java 1560 AB189849 Sida yellow vein Vietnam virus SiYVVNV Vietnam 2753 DQ641696 Mimosa yellow leaf curl virus MiYLCV Vietnam 2757 DQ641695 Malvastrum yellow vein virus-[Y47] MYVV China 2731 AJ457824 Malvastrum yellow vein Yunnan virus MYVVNV China 2747 AJ786711 NS, the sequence has not been submitted to GenBank Table 1 List of geminiviruses used for viral sequence analysis obtained from Asian Vegetable Research and D e v e l o p m e n t C e n t e r ( AV R D C ) , Ta i w a n . Amplification with PCR technique was carried out in a 25 µL reaction mixture containing 1 µL (200 n ) of sample DNA solution and 1 µL (0.2 µM) of each primer using Ready To Go PCR kit (Amersham Life Science). PCR was performed in thermalcycler Gen Amp PCR System9700 (Perkin Elmer) with 30 cycles of melting, annealing and DNA extension at 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 2 min, respectively. PCR products were then analysed by electrophoresis in 1% agarose gels in Tris-EDTA buffer (0.5x) and visualized under UV transilluminator (Maniatis . 1982). D N A f r a g m e n t s o f approximately 760 bp, as a product of PCR amplification, was sent to Macrogen Inc. (South Korea) for DNA sequencing by the dideoxy nucleotide chain termination method. Sequence data were then assembled and analyzed with the aid of Bioedit Programme and PAUP version 4.0 (Swofford 2002). sequences available from GenBank were used for phylogenetic analysisis (Table 1). The cladogram was set up with a quantitative cladistic maximum parsimony using heuristic methods. A hundred bootstrap iterations were performed. PCR using specific primers CP Protein-V1/CP Protein-C1 was successfully amplified a ~ 760 base pairs of coat protein fragments from 9 symptomatic samples (Fig 1) i.e. 5 samples from Bogor (AgrBgr), Sukabumi (AgrSkm), Magelang (AgrMgl), Sleman (AgrJgy), and Garut (AgrGrt); from Magelang (CtpMgl); from Sleman (AcpJgy); from Bogor g TM et al Geminivirus A. conyzoides C. minima A. boehmerioides P. ruderale S e q u e n c e a n a l y s i s . RESULTS Amplification and Sequencing of Coat Protein Gene. (PrlBgr); from Magelang (SplMgl). DNA fargments were not obtained from other weed samples ( from Garut from Brebes, from Garut, and from Cianjur). Major constraint for PCR-based detection using weed samples occurred on viral DNA extraction. Field samples tend to easily damage thus required immediate processing. In addition, inhibitor and secondary metabolites components found in weed tissues may inhibit the amplification process using PCR. Nucleotide sequence data was obtained from 7 virus samples (Table 1). Nucleotide's length of the virus that was successfully sequenced and used for sequence comparison was in the range of 756 to 993 bp, which contains parts of geminivirus coat protein gene (Santoso . 2008). Coat protein fragment analysis showed that weed-infecting geminiviruses collected from this study can be differentiated into 2 groups (Fig 2). The first group consists of 6 weed-infecting geminiviruses from this study (AgrBgr, AgrSkm, AgrJgy, AgrMgl, SplMgl, and CtpMgl) with a 100 bootstrap value, and the second group consists of one weed-infecting geminivirus from this study (PrlBgr) and other geminiviruses previously reported in GenBank with a 92 bootstrap value. Further more, the virus isolates in the first group can be differentiated into 4 subgroups each consisting of AgrBgr and AgrSkm, AgrJgy alone, AgrMgl and SplMgl, and CtpMgl alone. In the second group the virus isolate PrlBgr was placed in the different sub group with other weed-infecting geminiviruses. None of the weed infecting geminiviruses collected from this study has close relationship with geminiviruses previously reported from Java, Indonesia (TLCV, TLCV-[Ageratum], PepYLCV). S. iabadicensis G. parviflora , E. prostrata I. triloba L. peruviana et al Analysis of Genetic Relationship. 122 MELIANSYAH ET AL. Microbiol Indones Fig 1 Amplification product of geminiviral DNA fragment using specific primers CP Protein-V1/CP Protein-C1. The samples consist of weed-infecting geminiviruses : K. Positive control (artificially inoculated geminivirus from ); 1. -Bogor; 2. -Sukabumi; 3. -Magelang; 4. -Sleman; 5. -Garut; 6. -Magelang; 7. -Magelang; 8. -Brebes; 9. -Garut; 10. -Garut; 11. -Jogyakarta, 12; Cianjur; 13. -Bogor; M. 1 kb DNA A. conyzoides A. conyzoides A. conyzoides A. conyzoides A. conyzoides A. conyzoides S. iabadicencis C. minima E. prostrata G. parviflora I. triloba A. conyzoides L. peruviana- P. ruderale ladder. M K 21 3 4 5 6 7 8 9 1110 12 13 1000 bp 1750 bp 760 bp DISCUSSIONS Weeds are potential sources of primary inoculum of viruses and play an important role in their persistence and spread (Hallan . 1998). Weed infecting geminiviruses has been reported from different geographic location especially in the region where the geminivirus infection causing significant yield loss in important crops. Roye and McLaughlin (1997) reported a distinct geminivirus species from spp., , and when they studied the role of weed species in the establishment of tomato and pepper diseases due to infection in Jamaica. Artificial inoculation using involving several weed species was conducted by Sulandari . (2006) to determine the host range of pepper infecting geminivirus and concluded that , , , were very susceptible to PepYLCV infection. Yellow vein symptom or leaf netting is commonly found associated with geminivirus infection on weed species. During field survey to chilli pepper growing area in 2009, we easily found showing yellow vein symptom. Evidence of infection on et al Sida Macroptilium lathyroides Wissadula amplissima Geminivirus Bemisia tabaci et al H. brevipes P. floridana C. juncea A. conyzoides A. conyzoides Geminivirus A. Fig 2 Cladogram showing the interrelationship among weed-infecting geminiviruses and relative geminivirus' species based on allignment of part of coat protein gene's nucleotide sequence. Boothstrap value (100 replication) are shown on the branch of cladogram. Bean Yellow Dwarf Virus (BYDV) is included as outgroup. AgrBgr = Bogor, AgrSkm = Sukabumi, AgrMgl Magelang, AgrJgy= Sleman, SplMgl = BYDV = from South Africa (DQ458791), TLCV Java = T from Java, Indonesia (AB100304), TLCV Java = -[Ageratum] from Java, Indonesia (AB162141), TLCV Malaysia = T segment A from Malaysia (AF327436), TLCV Laos = from Malaysia (AF195782), AYVV Taiwan = from Taiwan (AF307861), AYVV China = from China (AJ495813), ChilCVA = -[Multan] from Pakistan (AF336806), PepLCV = segment A from Bangladesh (PAF314531), PepYLCV = P virus from West Java, Indonesia (AB189849), SiYVV = from Vietnam (SiYVV DQ641696), MiYLCV = from Vietnam (DQ641695), MYVV = from China (AJ457824), MYVYnV = from China (Aj786711). A. conyzoides- A. conyzoides- = A. conyzoides= A. conyzoides- S. iabadicencis-Magelang, CtpMgl =C. minima-Magelang, PrlBgr = P. ruderale-Bogor, Bean yellow dwarf virus omato leaf curl Java virus Tomato leaf curl Java virus omato leaf curl Malaysia virus Tomato leaf curl Laos virus Ageratum yellow vein Taiwan virus Ageratum yellow vein China virus Chili leaf curl virus Pepper leaf curl Bangladesh virus epper yellow leaf curl Indonesia Sida yellow vein virus Mimosa yellow leaf curl virus Malvastrum yellow vein virus Malvastrum yellow vein Yunnan virus conyzoides et al et al Geminivirus Geminivirus C. minima, P. ruderale, S. iabadicensis. Geminiviruses et al et al A. conyzoides C. minima, P. in Indonesia has been reported previously by Haerani and Hidayat (2003), Sukamto . (2005), and Kon . (2007). New infection on weed species in Indonesia was reported in this paper, i.e. infecting and Preliminary reports indicated that the primary infecting weeds are not the same ones that infect crops (Gilbertson . 1991; Mc Laughlin . 1994), although it has been speculated that a number of common weeds may serve as alternate hosts for crop-infecting geminiviruses. Based on phylogenetic analysis, Roye and McLaughlin (1997) concluded that weed-infecting geminiviruses are not host to crop- infecting geminiviruses in Jamaica. Similarly, phylogenetic relationships of the weed-infecting viruses collected in this study with other geminiviruses indicate that crop- and weed-infecting geminiviruses from Java, Indonesia are distinct, and highly diverse. Despite all seven weed-infecting geminiviruses were collected from endemic area of pepper yellow leaf curl disease in Java, none of them has a close relationship with PepYLCV Indonesia. Based on our phylogenetic analysis, it is evident that , Volume 5, 2011 Microbiol Indones 123 ruderale, S. iabadicensis Ludwigia peruviana A. conyzoides S. iabadicensis P. ruderale Synedrella nodiflora Galinsoga parviflora et al Tomato leaf curl Java virus Ageratum yellow vein virus and are not reservoirs for geminiviruses important on chilli pepper and tomato in Java. However, our host range study showed that PepYLCV was able to infect , , , , , and (data not published). Earlier, Kon . (2007) found evidence for interspecies recombination between (ToLCJV) and a strain of (AYVV - [Java]). Therefore, the importance of weeds as alternative hosts for crop- infecting geminiviruses in Indonesia will need further investigation. These results may significantly affect the development of strategies for managing the spread of these geminiviruses. This work was supported by the Australian Center for International Agricultural Research (Project No. HORT/2004/048), and Competitive Research Grant, Ministry of National Education, Republic of Indonesia (DIPA No. 0041/023-04.1/-/2010). We are grateful to Sri Sudarmiyati Tjitrosoedirdjo for her assistance in weed identification. ACKNOWLEDGEMENTS REFERENCES Fauquet CM, Stanley J. 2005. Revising the way we conceive and name viruses below the species level: a review of geminivirus taxonomy calls for new standardized isolate descriptors. Arch Virol. 150 (10):2151- 2179. doi:10.1007/s00705-005-0583-0. Gilbertson RL, Hidayat SH, Martinez RT, Leong SA, Faria JC, Morales F, Maxwell DP. 1991. Differentiation of bean infecting geminiviruses by nucleic acid hybridization probe and aspects of bean golden mosaic in Brazil. Plant Dis. 75(4):336-342. doi:10.1094/PD-75-0336. Haerani, Hidayat SH. 2003. 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Java virus Ageratum yellow vein virus Tomato leaf curl virus (Philippines) Begomovirus Sida Macropilium lathyroides Wissadula amplissima Begomovirus Begomovirus Geminivirus Begomovirus Capsicum annuum Malvastrum leaf curl Guangdong virus Begomovirus : Begomovirus Emilia sonchifolia. 124 MELIANSYAH ET AL. Microbiol Indones