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FIRST REPORT of STEM CANKER DISEASE on Acacia Mangium INDUCED by Lasiodiplodia Theobromae and Lasiodiplodia Pseudotheobromae SPECIES in SABAH, MALAYSIA

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FIRST REPORT of STEM CANKER DISEASE on Acacia Mangium INDUCED by Lasiodiplodia Theobromae and Lasiodiplodia Pseudotheobromae SPECIES in SABAH, MALAYSIA Malays. Appl. Biol. (2018) 47(3): 147–151 FIRST REPORT OF STEM CANKER DISEASE ON Acacia mangium INDUCED BY Lasiodiplodia theobromae AND Lasiodiplodia pseudotheobromae SPECIES IN SABAH, MALAYSIA MANDY MAID1,2, AZMAH A. LATIF1, EDMUND GAN3, NAJJAH SALFINAS4, CRISPIN KITINGAN5, COLIN R. MAYCOCK2 and WICKNESWARI RATNAM1* 1School of Environmental and Natural Resource Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor Darul Ehsan, Malaysia 2Faculty of Science and Natural Resources, Universiti Malaysia Sabah, Jalan UMS, 88400 Kota Kinabalu, Sabah, Malaysia 3Sabah Forest Industries Sdn. Bhd., S.F.I. Complex, No. 10, Jalan Jeti, W.D.T. 31, 89859 Sipitang, Sabah 4Acacia Forest Industries, Acacia Forest Industries Sdn. Bhd., Level 2, Wisma Perkasa, Jalan Gaya, P.O. BOX 17553, 88873 Kota Kinabalu, Sabah 5Sabah Forestry Development Authority, Locked Bag No. 122, 88999 Kota Kinabalu, Sabah *E-mail: [email protected] Accepted 3 May 2018, Published online 30 June 2018 Acacia mangium is an important timber plantation grey then darkened with age at 5 days, 10 days and crop planted mainly in the eastern states of Malaysia 30 days after plating (Figure 2a-c, 3a-c). The plates over the last three decades. In a field disease survey were filled with compact dark mycelium within a conducted from April to June 2013, cankers were week. Lasidiploida theobromae had whitish tendrils detected on the stems of planted A. mangium trees on a slight dark bulge (Figure 2c), whilst L. aged one to seven years old at three locations pseudotheobromae had white to light grey tufts on (Bongkol, Pitas (6°59’00"N, 117°1’00"E); Kota the fungal colony (Figure 3c). Conidia was observed Belud, West Coast (6°30’30"N, 116°34’40"E); in 30-days and older cultures. Immature conidia were Sipitang, Interior Division (Padas 5°2’00"N, unicellular, aseptate, and hyaline (Figure 2d, 3e). 115°37’00"E and Ganui, 4°50’00"N, 115°42’00"E) Mature conidia were brownish, longitudinally in Sabah, Malaysia (Maid & Wickneswari, 2014). striate, and two-celled with one septum and Infected area of the stems had slight swelling and thickened wall (Figure 2e, 3e). The average size necrotic lesions that were dark brown with intense of a conidium was 13.6 x 26.3 μm (n=20) and 14.8 colouring in the centre. In severe cankers, the bark x 29.4 μm (n=20) for L. theobromae and L. breaks up in irregular strips along the branches and pseudotheobromae respectively. Total genomic stems with brownish exudate beneath the bark and DNA was extracted from 10-day old cultures, and from the wounds. The sapwood was discoloured the internal transcribed spacer (ITS) region was (light brown to nearly black) and often rotten amplified using the primers ITS1/ITS4 (White et al., beneath the necrotic lesions and had fractured bark 1990) and sequenced according to Van Wyk et al. (Figure 1). A total of 26 canker infected trees were (2006). The sequences of canker isolates SSB2A, sampled from all locations. Small pieces (5-10mm) AKB4T5 and GL28B44T10 sequences were of infected wood were surface sterilized for 1 min deposited to GenBank (Accessions Nos. KP998515, with 2% sodium hypochlorite and rinsed 3 times KP998517 and KP998522 respectively). The with sterile distilled water, air-dried on filter sequences had 99 to 100% identity to L. theobromae paper and plated onto 2% potato dextrose agar reference sequences in GenBank database (PDA) supplemented with streptomycin sulphate (Accession Nos. KC964548.1, EF622073.1, (0.001g/L-1). Pure cultures were obtained by hyphal JX982240.1 respectively). The sequence of canker tip excision from the margins of the colony plated isolate SSB2B (Accessions no. KP998516) had on 2% PDA and incubated at 25°C. Fungal cultures 99% identity to Lasiodiplodia pseudotheobromae initially produced white mycelium which turned (Accession no. EU860391.1). Figure 4 is the phylogenetic tree constructed using the nucleotide * To whom correspondence should be addressed. sequences of the fungal isolates on the web service 148 STEM CANKER DISEASE ON Acacia mangium Fig. 1. Canker symptoms indicated by arrows on 4 year old Acacia mangium trees. Fig. 2. Lasiodiplodia theobromae. (a-c) Young to older colonies darken with age at 5 days, 10 days, and 30 days (c) white spore tendrils (indicated by arrow) containing young conidia appear on dark mycelium, (d) young and immature conidia, (e) mature conidia. Fig. 3. Lasiodiplodia pseudotheobromae (a-c) Young to older colonies darkening with age at 10 days, 15 days, and 30 days (a, c) white to grey tufts on the colony, (d) conidiogenous cells, (e) young and mature conidia. Fig. 4. Phylogenetic tree of fungal isolates used in pathogenicity test. STEM CANKER DISEASE ON Acacia mangium 149 Fig. 5. Pathogenecity test on A. mangium. (a) Healthy A. mangium phyllode, (b) Lesion indicated by arrow on phyllode inoculated with L. theobromae after 10 days, (c) Lesion indicated by arrow on phyllode inoculated with L. pseudotheobromae after 10 days, (d) Control seedling inoculated with PDA plug remains healthy and free of lesion, (e) Seedling inoculated with L. theobromae show slight drying of the phyllodes, (f) Seedling inoculated with L. pseudotheobromae wilt and defoliate, (g) Cross-section of A. mangium seedling stem inoculated with L. theobromae with lesion and necrotic tissue, (h) Cross-section of A. mangium seedling stem inoculated with L. pseudotheobromae with lesion and necrotic tissue. Phylogeny.fr (http://www.phylogeny.fr/index.cgi) caused by L. theobromae measured 3.8cm and (Deereper et al., 2008, 2010) showing the isolate 1.2cm compared to average lesion size caused by GL28B44T10 (L. theobromae) set apart. Patho- L. pseudotheobromae was 5.3cm and 3.8cm on the genicity test was carried out by inoculating 7 A. mangium phyllodes and seedlings. Both fungi months-old Acacia mangium seedlings using L. were re-isolated from the diseased phyllodes and theobromae isolate SSB2A in a greenhouse seedlings to complete Koch’s postulates. experiment; and inoculation of detached phyllodes Lasiodiplodia theobromae affects a wide range of in the laboratory. A PDA agar plug obtained from agronomically important plant hosts (Del Castillo the margins of 10-day old fungal isolate was placed et al., 2016; Haggag, 2014; Sulaiman et al., 2012) on fresh wounds made on the bark of the seedlings and forest trees (Borges et al., 2015; Kausar et al., (5mm) and on the primary veins of the phyllode 2009) in the tropics. Ever since L. pseudo- (2mm). Nine A. mangium seedlings and fifteen theobromae was first described (Alves et al., 2008) detached phyllodes were inoculated with agar plugs it has been reported as a pathogen in the tropic and from SSB2A, SSB2B isolates and PDA plugs sub-tropic hosts (Dissanayake et al., 2015; Ismail et (control) respectively. The inoculation wound was al., 2012; Kwon et al., 2017; Nogueira Júnior et al., moistened and bound with parafilm on the seedlings 2017; Tennakoon et al., 2016; Zhao et al., 2010) and cellophane tape for the phyllode. After 10 days, including planted A. mangium (Castro-Medina et al., A. mangium seedlings and detached phyllodes 2013). Both species are the most common canker inoculated with fungus showed symptoms of lesion and rot causing fungus in the genus Lasiodiplodia and necrotic tissue, while the control did not show (Coutinho et al., 2017; Netto et al., 2017). To our disease symptoms (Figure 5). Average size of lesion knowledge, this is the first report of L. theobromae 150 STEM CANKER DISEASE ON Acacia mangium (Pat.) Griffon and Maubl. and L. pseudotheobromae Dissanayake, A.J., Zhang, W., Mei, L., Chukeatirote, sp. nov. as virulent pathogens causing stem canker E., Yan, J.Y., Li, X. & Hyde, K.D. 2015. in A. mangium in Sabah, Malaysia. Lasiodiplodia pseudotheobromae causes pedicel and peduncle discolouration of grapes in China. Australasian Plant Disease Notes, 10: ACKNOWLEDGEMENTS 21. Dereeper, A., Audic, S., Claverie, J.M. & Blanc, G. The project was funded by Ministry of Science and 2010. BLAST-EXPLORER helps you building Technology (MOSTI), Malaysia (02-01-02-SF0765). datasets for phylogenetic analysis. BMC SLAI scholarship for Mandy Maid was funded by Evoloution Biology, 12: 10-8. Ministry of Education (MOE), Malaysia and Dereeper, A., Guignon, V., Blanc, G., Audic, S., Universiti Malaysia Sabah. We thank Acacia Forest Buffet, S., Chevenet, F., Dufayard, J.F., Guindon, Industry Sdn. Bhd. (AFI), Sabah Forest Industry Sdn. S., Lefort, V., Lescot, M., Claverie, J.M. & Bhd. (SFI), and Sabah Forestry Development Gascuel, O. 2008. Phylogeny.fr: robust phylo- Authority (SAFODA) for technical assistance, access genetic analysis for the non-specialist. Nucleic to study sites and Acacia seeds; Assoc. Prof. Dr. Acids Research, 1: 36. Choong Chee Yen for assisting with DNA sequence Haggag, W.M. 2014. First report of Lasiodiplodia analysis; Nur Qayrani Mohamed for conducting theobromae causing canker and collar rot fungal pathogenicity testing. diseases of physic nut (Jatropha curcas) in Egypt. Journal of Plant Pathology, 96(3): 603- 611. REFERENCES Ismail, A.M., Cirvilleri, G., Polizzi, G., Crous, P.W., Groenewald, J.Z. & Lombard, L. 2012. Alves, A., Crous, P.W., Correia, A. & Phillips, A.J.L. Lasiodiplodia species associated with dieback 2008. Morphological and molecular data reveal disease of mango (Mangifera indica) in Egypt. cryptic speciation in Lasiodiplodia theobromae. Australasian Plant Pathology. DOI 10.1007/ Fungal Diversity, 28: 1-13. s13313-012-0163-1. Borges, R.C.F., Santos, M.D.M., Macedo, M.A., Kausar, P., Chohan, S. & Parveen, R. 2009. Physio- Martins, I., Nascimento, A.G., Café-Filho, A.C., logical studies on Lasiodiplodia theobromae Boiteux, L.S., Fonseca, M.E.N., Inácio, C.A. & and Fusarium solani, the cause of Shesham Mello, S.C.M. 2015. A trunk canker disease of decline.
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