Accepted Manuscript

Title: Novel mastreviruses identified in Australian wild rice

Authors: Simona Kraberger, Andrew D.W. Geering, Matthew Walters, Darren P. Martin, Arvind Varsani

PII: S0168-1702(17)30390-8 DOI: http://dx.doi.org/doi:10.1016/j.virusres.2017.07.003 Reference: 97188

To appear in: Virus Research

Received date: 19-5-2017 Revised date: 29-6-2017 Accepted date: 2-7-2017

Please cite this article as: Kraberger, Simona, Geering, Andrew D.W., Walters, Matthew, Martin, Darren P., Varsani, Arvind, Novel mastreviruses identified in Australian wild rice.Virus Research http://dx.doi.org/10.1016/j.virusres.2017.07.003

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Novel mastreviruses identified in Australian wild rice

Simona Kraberger1, Andrew D.W. Geering2*, Matthew Walters3, Darren P. Martin4 and Arvind Varsani1,5*

1 The Biodesign Center for Fundamental and Applied Microbiomics, Center for Evolution and Medicine, School of Life sciences, Arizona State University, Tempe, AZ 85287-5001, USA

2 Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, 41 Boggo Road, Dutton Park, QLD 4102, Australia

3 School of Biological Sciences, University of Canterbury, Christchurch 8140, New Zealand

4 Computational Biology Group, Institute of Infectious Diseases and Molecular Medicine, University of Cape Town, Cape Town, South Africa

5 Structural Biology Research Unit, Department of Clinical Laboratory Sciences, University of Cape Town, Observatory, Cape Town, South Africa

Keywords: Geminivirus, Oryza sp. ‘Taxon A’, ‘Taxon B’, Oryza australiensis.

*Corresponding authors

Dr Andrew Geering: [email protected]

Dr Arvind Varsani: [email protected]

Abstract: 156 words Text: 1674 words Figures: 2 Table: 1 Supplementary Data: 1

Hightlights  First report of mastreviruses infecting Oryza sp.  Two novel mastreviruses species identified infecting wild rice  22 genomes of rice latent virus 1 from nine sampling sites  One genome rice latent virus 2 from one sampling site

Abstract

Most known mastreviruses (family ) infect members of the grass family, Poaceae. Although the greatest number of grass-infecting mastrevirus species have been discovered in Africa, it is apparent that the ten grass-infecting mastrevirus species that have so far only been discovered in south-east Queensland have a degree of diversity that rivals that observed in Africa. In this study, we have used a deep sequencing approach to identify two new mastrevirus species, tentatively named rice latent virus 1 and 2 (RLV 1 and 2), from two, undescribed wild rice species (Oryza AA genome group) in Cape York Peninsula, Queensland. The sequences of these new had less than 70% identity with any previously identified mastrevirus, and therefore their discovery vastly expands the known diversity of monocot-infecting mastreviruses in Australia. This study also highlights the potential risks of novel crop pathogens emerging from uncultivated grass species, as the wild rice hosts are very closely related to domesticated rice.

Various mastreviruses ( Mastrevirus, family Geminiviridae) are pathogens of economically important crops, most notably maize in Africa and chickpea in the Middle East. Thirty-seven mastrevirus species have been documented from Africa, the Americas Asia, Australia Europe, and various Indian and Pacific Ocean islands. Seven of these known species infect dicotyledonous plants and 30 species infect monocotyledonous plants (Agindotan et al., 2015; Candresse et al., 2014; Kraberger et al., 2015; Muhire et al., 2013; Oluwafemi et al., 2014; Zerbini et al., 2017). The monocot-infecting mastreviruses are the most widely distributed, genetically diverse group, and infect both cultivated and wild members of the Poaceae (Kraberger et al., 2013; Muhire et al., 2013; Oluwafemi et al., 2014; Varsani et al., 2009). Africa and Australia have been identified as global diversity hotspots for mastreviruses (Kraberger et al., 2013; Kraberger et al., 2012; Varsani et al., 2008), containing about 65% of the known species.

Rice (Oryza sativa) is a staple crop for a large proportion of the global population with more than 480 million metric tons being produced annually (Muthayya et al., 2014). The genus Oryza comprises 22–24 species, which are pantropical in distribution and divided into six diploid (A-C and E-G) and five tetraploid (BC, CD, HJ, HK and KL) genome groups, based on molecular systematics and reproductive compatibility (Ammiraju et al., 2010; Ge et al., 1999; Lu et al., 2009). O. sativa, and the only other cultivated rice species, O. glaberrima, are both classified as having an AA genome. Australia is a center of diversity and the possible origin of the AA genome group (Brozynska et al., 2017). There is considerable scientific interest in the wild species because of their potential to be used in rice breeding programs (Henry et al., 2010) but also because of the risks they pose to rice production by acting as reservoirs of pests and pathogens (Khemmuk et al., 2016; Petrovic et al., 2013). The fungus Pyricularia oryzae, the cause of blast disease, is thought to have jumped from wild to domesticated rice in north Queensland, an event that has led to the temporary abandonment of cropping in parts of Cape York Peninsula until disease management strategies can be devised (Khemmuk et al., 2016).

Surveys for monocot-infecting mastreviruses have predominantly been in south-east Queensland, close to the major urban centre, Brisbane. The vast monsoonal grasslands of northern Australia have never been explored for mastreviruses, mainly due to logistical reasons. The wild rice species found in Cape York Peninsula, Queensland (Oryza australiensis (EE genome) and two undescribed AA genome-type species, Oryza sp. Taxon A and Oryza sp. Taxon B), grow on flood plains and the fringes of lagoons, and are perennial, dying back to buried rhizomes during the dry season (Henry et al., 2010). Many perennial monocots, such as sugarcane and banana, support large assemblages of plant viruses, probably due to the fact that infections persist from one generation to the next via vegetative propagation of the plants e.g. potyviruses (Xu et al., 2008). In this study, we hypothesized that the wild rice species in Queensland would likewise be hosts of plant viruses, especially mastreviruses, given the long presence of this group of viruses in Australia.

During 12–14 May 2014 and 27–29 March 2015, wild rice plants at various sites (n=11) in Cape York Peninsula, from the Atherton Tableland to Rinyirru (Lakefield) National Park (Figure 1; Table 1), were sampled for testing for leaf spot pathogens (see Khemmuk et al. (2016)) and viruses (this study). Genomic DNA extracts from 58 wild rice plants (Table 1) were prepared using a GF- 1 Nucleic Acid Extraction Kit (Vivantis Technologies, Malaysia). One µl of genomic DNA from each sample was used to amplify circular DNA by rolling circle amplification using an Illustra TempliPhi Amplification Kit (GE Healthcare, USA). The RCA products were pooled (1 µl from each amplification) and sequenced on an Ilumina Hiseq 2500 platform at Macrogen Inc (Korea). Resulting paired-end reads were de novo assembled using ABySS 1.9 (Simpson et al., 2009) and contigs >1000 nt were analysed against a local viral database using BLASTX (Altschul et al., 1990). Two contigs (~2.7 kb) with potentially encoded proteins with detectable similarity to those of mastreviruses were identified. Pairs of abutting primers were designed (RLV-1F: 5’- CCC ACC GCA CAA TAC AAT GTT ATA GAC-3’ and RLV-1R: 5’- GTC ATA AGT GGT GAA GTC CAC CAG-3’; RLV-2F: 3’-TGT GTT TCT GAG CTT CAT AAA GAT GGG-3’ and RLV-2R: 5’- TAA AAC GCC GTA AGG CCT GAG CAG A-3’) to screen and recover complete genomes of these mastreviruses from each of the wild rice samples. PCR was done using HiFi hotstart DNA polymerase (Kapa Biosystems, USA) and 1 µl of RCA products as a template in a 20 µl total volume. Thermal cycling conditions were: 94 ºC for 3 min, 25 cycles of 98 ºC (20 s), 55 ºC (30 s), 72 ºC (3 min) with a final extension of 72 C for 3 min. PCR products were resolved on a 0.7% agarose gel and fragments ~2.7 kb in length excised and gel purified. The DNA was then cloned in pJET1.2 plasmid (Thermo Fisher Scientific, USA) and Sanger sequenced (Macrogen Inc., Korea) by primer walking. From 22 of the 58 wild rice samples, mastrevirus genomes were recovered using primer pair RLV-1 F/R and from one additional sample another genome was recovered using primer pair RLV-2 F/R (Table 1).

Viral sequences were assembled using DNA Baser (Heracle BioSoft S.R.L. Romania) and open reading frames identified using DNAMAN V7 (Lynnon Biosoft, Canada). Datasets including species and strains from all previously identified mastreviruses (downloaded on 25th April 2017) were compiled in MEGA V7 (Kumar et al., 2016), aligned using MUSCLE (Edgar, 2004) and a full genome neighbor-joining phylogenetic tree was constructed using Jukes-Cantor substitution model, 1000 bootstrap replicates, and rooted with beet curly top Iran virus (Figure 2). Maximum likelihood phylogenetic trees were constructed for CP and Rep amino acid sequence datasets using PhyML3 (Guindon et al., 2010) implemented in Seaview V4 (Gouy et al., 2010) with aLRT support, and Eragrostis curvula streak virus to root the trees. Best fit models for CP (LG+I+G+F) and Rep (LG+I+G) were identified using ProtTest (Darriba et al., 2011) (Figure 2).

Pairwise comparison of the full genome dataset using SDT v1.2 (Muhire et al., 2014) indicated that all new sequences had less than 70% pairwise identity with all other known mastreviruses (Supplementary Data 1). Following the guidelines for mastrevirus species demarcation (i.e. a species demarcation threshold of 78 % coupled with phylogenetic support (Muhire et al., 2013), two novel mastrevirus species were identified, one represented by 22 virus isolates and the other, by a single isolate. No wild rice specimen had overt disease symptoms such as foliar chlorotic mosaics or streaking and therefore the two new mastrevirus species were tentatively named rice latent virus 1 (n=22) and 2 (n=1) (RLV 1 and 2). RLV 1 was detected in both Oryza sp. ‘Taxon A’ and ‘B’ but not O. australiensis, although only a single location containing the latter was surveyed. RLV 1 was very common and widely distributed, as it was found at nearly every site that was visited. However, RLV 2 was only found at one site, Red Lily Lagoon in Rinyirru National Plant, in a single plant of Oryza sp. ‘Taxon B’.

The 22 RLV 1 genomes shared >98% identity with one another and ranged in size from 2754-2758 nt. The single RLV 2 genome is 2843 nt in length. Both viruses had typical mastrevirus genome architecture with genes encoding a movement protein (MP) and a protein (CP) on the virion- sense strand and a RepA and a replication associated protein (Rep) on the complementary sense- strand. The nonanucleotide motif at the presumed origin of virion-strand replication in RLV 2 is identical to that of most other monocot-infecting mastreviruses (TAATATTAC), whereas RLV 1 has a TAATGTTAC sequence that has only previously been observed in the genomes of some dicot-infecting mastreviruses and a mastrevirus-like genome sequence recovered from a dragonfly in Puerto Rico (dragonfly-associated mastrevirus; JX458740; (Rosario et al., 2013).

Phylogenetic analysis of the full genomes of the new rice-infecting mastreviruses showed that RLV 1 is most closely related to Eragrostis minor streak virus (EMSV; JF508490; from Namibia) and Miscanthus streak virus (MiSV; E02258; from Japan), with which it shares between 60 and 62% genome-wide pairwise nucleotide identity. RLV 2 is most closely related to Axonopus compressus streak virus (ACSV; KJ437671; from Nigeria) with which it shares 70% pairwise identity (Figure 2A). In phylogenetic analyses using the putative Rep and CP amino acid sequences, RLV 2 consistently clustered with ACSV (Figure 2B). The putative CP of RLV 1 is most closely related to the CPs of EMSV and MiSV (with which it shares between 42 and 45% pairwise amino acid sequence identity), whereas the putative Rep of RLV 1 clusters with those of , oat dwarf virus, dicot-infecting mastreviruses, MiSV and EMSV with which it shares between 47 and 55% pairwise amino acid sequence identity (Figure 2B; supplementary data 1).

This is the first report of mastreviruses identified infecting Oryza sp. The low degrees of genetic relatedness between wild rice-infecting and other known mastreviruses further emphasises the fact that only a portion of the total mastrevirus diversity has been uncovered. In the last few years, known and unknown viruses have been identified in regions where mastreviruses were previously not thought to exist e.g. wheat dwarf India virus in India, maize streak Reunion virus in China, sugarcane white streak virus in Barbados, sugarcane striate virus in China, oat dwarf virus in Iran and switchgrass mosaic associated virus in the USA (Agindotan et al., 2015; Candresse et al., 2014; Chen et al., 2015; Kamali et al., 2017; Kumar et al., 2012). While the ongoing discovery of novel mastreviruses within both cultivated grasses and their uncultivated relatives provides the basis of mapping the past and ongoing colonization of the world by these viruses, it is also an essential first step in identifying instances where these viruses are in the process of emerging as pathogens of economic significance.

Acknowledgments DPM and AV are supported by the National Research Foundation of South Africa. Partial support for the surveys was provided by the Plant Biosecurity CRC as part of the Australian Government’s Cooperative Research Centres Program.

Figure 1: Location of wild rice sampling sites in the Cape York Peninsula, Queensland, Australia. Fifty-three samples were collected from 11 different sites (see Table 1 for sample and site details). Rice latent virus 1 was identified in samples from ten sites (1-10) where either Oryza sp. ‘Taxon A’ or ‘Taxon B’ was growing. Rice latent virus 1 was only identified at site 5. At site 11, Oryza australiensis was present and this was the only site where no mastreviruses were detected.

Figure 2: (A) Neighbor-joining phylogenetic tree of full genomes, and (B) Maximum likelihood phylogenetic trees of the replication associated protein (Rep) and capsid protein (CP) amino acid sequences with representative sequences from all known mastrevirus species and strains together with those newly identified (bold font).

Supplementary Data 1: Pairwise comparisons of the full genome nucleotide, and Rep and CP amino acid sequences of representative mastrevirus (species and strains) with those recovered in this study (names highlighted in red).

GenBank accession #s: KY962359 - KY962381

References Agindotan, B.O., Domier, L.L., Bradley, C.A., 2015. Detection and characterization of the first North American mastrevirus in switchgrass. Arch Virol 160(5), 1313-1317. Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J Mol Biol 215(3), 403-410. Ammiraju, J.S., Fan, C., Yu, Y., Song, X., Cranston, K.A., Pontaroli, A.C., Lu, F., Sanyal, A., Jiang, N., Rambo, T., Currie, J., Collura, K., Talag, J., Bennetzen, J.L., Chen, M., Jackson, S., Wing, R.A., 2010. Spatio-temporal patterns of genome evolution in allotetraploid species of the genus Oryza. Plant J 63(3), 430-442. Brozynska, M., Copetti, D., Furtado, A., Wing, R.A., Crayn, D., Fox, G., Ishikawa, R., Henry, R.J., 2017. Sequencing of Australian wild rice genomes reveals ancestral relationships with domesticated rice. Plant Biotechnol J 15(6), 765-774. Candresse, T., Filloux, D., Muhire, B., Julian, C., Galzi, S., Fort, G., Bernardo, P., Daugrois, J.- H., Fernandez, E., Martin, D.P., Varsani, A., Roumagnac, P., 2014. Appearances can be deceptive: Revealing a hidden viral infection with deep sequencing in a plant quarantine context. PLoS ONE 9(7), e102945. Chen, S., Huang, Q., Wu, L., Qian, Y., 2015. Identification and characterization of a maize- associated mastrevirus in China by deep sequencing small RNA populations. Virol J 12, e156. Darriba, D., Taboada, G.L., Doallo, R., Posada, D., 2011. ProtTest 3: fast selection of best-fit models of protein evolution. Bioinformatics 27(8), 1164-1165. Edgar, R.C., 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32(5), 1792-1797. Ge, S., Sang, T., Lu, B.R., Hong, D.Y., 1999. Phylogeny of rice genomes with emphasis on origins of allotetraploid species. Proc Natl Acad Sci U S A 96(25), 14400-14405. Gouy, M., Guindon, S., Gascuel, O., 2010. SeaView Version 4: A Multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Molecular Biology and Evolution 27(2), 221-224. Guindon, S., Dufayard, J.-F., Lefort, V., Anisimova, M., Hordijk, W., Gascuel, O., 2010. New Algorithms and Methods to Estimate Maximum-Likelihood Phylogenies: Assessing the Performance of PhyML 3.0. Systematic Biology 59(3), 307-321. Henry, R.J., Rice, N., Waters, D.L.E., Kasem, S., Ishikawa, R., Hao, Y., Dillon, S., Crayn, D., Wing, R., Vaughan, D., 2010. Australian Oryza: Utility and Conservation. Rice 3(4), 235- 241. Kamali, M., Heydarnejad, J., Pouramini, N., Masumi, H., Farkas, K., Kraberger, S., Varsani, A., 2017. Genome sequences of Beet curly top Iran virus, Oat dwarf virus, Turnip curly top virus, and Wheat dwarf virus Identified in . Genome Announcements 5(8), e01674_01616. Khemmuk, W., Shivas, R.G., Henry, R.J., Geering, A.D.W., 2016. Fungi associated with foliar diseases of wild and cultivated rice (Oryza spp.) in northern Queensland. Australas Plant Path 45(3), 297-308. Kraberger, S., Harkins, G.W., Kumari, S.G., Thomas, J.E., Schwinghamer, M.W., Sharman, M., Collings, D.A., Briddon, R.W., Martin, D.P., Varsani, A., 2013. Evidence that dicot- infecting mastreviruses are particularly prone to inter-species recombination and have likely been circulating in Australia for longer than in Africa and the Middle East. Virology 444(1–2), 282-291. Kraberger, S., Mumtaz, H., Claverie, S., Martin, D.P., Briddon, R.W., Varsani, A., 2015. Identification of an Australian-like dicot-infecting mastrevirus in Pakistan. Arch Virol 160(3), 825-830. Kraberger, S., Thomas, J.E., Geering, A.D.W., Dayaram, A., Stainton, D., Hadfield, J., Walters, M., Parmenter, K.S., van Brunschot, S., Collings, D.A., Martin, D.P., Varsani, A., 2012. Australian monocot-infecting mastrevirus diversity rivals that in Africa. Virus Research 169(1), 127-136. Kumar, J., Singh, S.P., Kumar, J., Tuli, R., 2012. A novel mastrevirus infecting wheat in India. Arch Virol 157(10), 2031-2034. Kumar, S., Stecher, G., Tamura, K., 2016. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution 33(7), 1870-1874. Lu, F., Ammiraju, J.S., Sanyal, A., Zhang, S., Song, R., Chen, J., Li, G., Sui, Y., Song, X., Cheng, Z., de Oliveira, A.C., Bennetzen, J.L., Jackson, S.A., Wing, R.A., Chen, M., 2009. Comparative sequence analysis of MONOCULM1-orthologous regions in 14 Oryza genomes. Proc Natl Acad Sci U S A 106(6), 2071-2076. Muhire, B., Martin, D.P., Brown, J.K., Navas-Castillo, J., Moriones, E., Zerbini, M.F., Rivera- Bustamante, R.F., Malathi, V.G., Briddon, R.W., Varsani, A., 2013. A genome-wide pairwise-identity-based proposal for the classification of viruses in the genus Mastrevirus (family Geminiviridae). Arch Virol 158(6), 1411-1424. Muhire, B.M., Varsani, A., Martin, D.P., 2014. SDT: A tool based on pairwise sequence alignment and identity calculation. PLoS ONE 9(9), e108277. Muthayya, S., Sugimoto, J.D., Montgomery, S., Maberly, G.F., 2014. An overview of global rice production, supply, trade, and consumption. Annals of the New York Academy of Sciences 1324(1), 7-14. Oluwafemi, S., Kraberger, S., Shepherd, D.N., Martin, D.P., Varsani, A., 2014. A high degree of African streak virus diversity within Nigerian maize fields includes a new mastrevirus from Axonopus compressus. Arch Virol 159(10), 2765-2770. Petrovic, T., Burgess, L.W., Cowie, I., Warren, R.A., Harvey, P.R., 2013. Diversity and fertility of Fusarium sacchari from wild rice (Oryza australiensis) in Northern Australia, and pathogenicity tests with wild rice, rice, sorghum and maize. Eur J Plant Pathol 136(4), 773- 788. Rosario, K., Padilla-Rodriguez, M., Kraberger, S., Stainton, D., Martin, D.P., Breitbart, M., Varsani, A., 2013. Discovery of a novel mastrevirus and alphasatellite-like circular DNA in dragonflies (Epiprocta) from Puerto Rico. Virus Research 171(1), 231-237. Simpson, J.T., Wong, K., Jackman, S.D., Schein, J.E., Jones, S.J., Birol, I., 2009. ABySS: a parallel assembler for short read sequence data. Genome Res 19(6), 1117-1123. Varsani, A., Shepherd, D.N., Dent, K., Monjane, A.L., Rybicki, E.P., Martin, D.P., 2009. A highly divergent South African geminivirus species illuminates the ancient evolutionary history of this family. Virology Journal 6, e36. Varsani, A., Shepherd, D.N., Monjane, A.L., Owor, B.E., Erdmann, J.B., Rybicki, E.P., Peterschmitt, M., Briddon, R.W., Markham, P.G., Oluwafemi, S., Windram, O.P., Lefeuvre, P., Lett, J.M., Martin, D.P., 2008. Recombination, decreased host specificity and increased mobility may have driven the emergence of as an agricultural pathogen. Journal of General Virology 89(9), 2063-2074. Xu, D.L., Park, J.W., Mirkov, T.E., Zhou, G.H., 2008. Viruses causing mosaic disease in sugarcane and their genetic diversity in southern China. Arch Virol 153(6), 1031-1039. Zerbini, F.M., Briddon, R.W., Idris, A., Martin, D.P., Moriones, E., Navas-Castillo, J., Rivera- Bustamante, R., Roumagnac, P., Varsani, A., Consortium, I.R., 2017. ICTV Virus Taxonomy Profile: Geminiviridae. Journal of General Virology 98(2), 131-133.

Table 1: Plant samples and virus isolates collected during surveys of Cape York Peninsula, Queensland, Australia.

Sample # Virus GenBank Site # Date Location Lat, Long Taxon accession NA1 – - 1 12 May Abattoir Swamp -16.63588, Oryza sp. ‘Taxon A’ NA2 – - 2014 Environmental Park, 145.32697 NA3 – - Julatten NA4 – - NA5 – - NA67 RLV 1 KY962377 NA68 RLV 1 KY962378 NA69 RLV 1 KY962379 NA70 RLV 1 KY962380 NA71 – - NA72 – - NA73 – - NA74 – - NA75 – - NA76 – - NA15 RLV 1 KY962359 2 13 May Unnamed Road, -14.99145, Oryza sp. ‘Taxon B’ 2014 Rinyirru National Park 144.26318

NA16 RLV 1 KY962360 3 13 May -14.97033, Oryza sp. ‘Taxon B’ 2014 144.24205

NA18 RLV 1 KY962361 4 13 May White Lily Lagoon, -14.86297, Oryza sp. ‘Taxon B’ NA19 – - 2014 Rinyirru National Park 144.16751 NA20 – - NA22 RLV 1 KY962362 5 13 May Red Lily Lagoon, -14.84953, Oryza sp. ‘Taxon B’ NA23 – - 2014 Rinyirru National Park 144.16817 NA24 RLV 2 KY962381 NA29 – - 6 13 May Near Sandy Creek, -15.53071, Oryza sp. ‘Taxon B’ NA30 RLV 1 KY962364 2014 Peninsula 144.38297 Development Road NA26 RLV 1 KY962363 7 13 May Billabongs north of -15.43943, Oryza sp. ‘Taxon B’ 2014 Laura 144.21114 NA27 – - 8 -15.46935, NA28 – - 144.24257 NA32 – - 9 14 May Pandanus Lagoon, -16.93790, Oryza sp. ‘Taxon B’ NA33 – - 2014 Mareeba Wetlands 145.35052 NA48 – - 10 28 Unnamed dam, -15.69936, Oryza sp. ‘Taxon A’ NA49 RLV 1 KY962365 March Mulligan Highway 145.02981 NA50 – - 2015 NA51 – - NA52 RLV 1 KY962366 NA53 RLV 1 KY962367 NA54 – - NA55 RLV 1 KY962368 NA56 RLV 1 KY962369 NA57 RLV 1 KY962370 NA58 – - NA59 RLV 1 KY962371 NA60 RLV 1 KY962372 NA61 – - NA62 RLV 1 KY962373 NA63 RLV 1 KY962374 NA64 RLV 1 KY962375 NA65 RLV 1 KY962376 NA87 – - 11 28 Ditch north of -15.87246, Oryza australiensis NA88 – - March entrance to Lakeland 144.860644 NA89 – - 2015 Downs power NA90 – - substation NA91 – - NA92 – - NA93 – - NA94 – - NA95 – - NA96 – -