Characterisation and Pathogenicity of Pyricularia oryzae from Different Regions of Sarawak

Elissa S., Rafael (2024) Characterisation and Pathogenicity of Pyricularia oryzae from Different Regions of Sarawak. Masters thesis, Universiti Malaysia Sarawak.

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Abstract

Rice blast disease (causal agent: Pyricularia oryzae) is an important rice disease in Sarawak. However, crucial information on P. oryzae of Sarawak such as genetic diversity, variation of avirulence (Avr) genes, resistance of Sarawak rice towards P. oryzae and potential alternative host for P. oryzae of Sarawak are still lacking. This study aims to characterise P. oryzae collected from rice and weed host from rice field in Kuching, Serian, Sri Aman, Betong and Sibu divisions of Sarawak, to assess the sequence variation of three Avr genes; AvrPiz-t, Avr-Pik and AvrPi-54 in P. oryzae isolates collected from Sarawak rice field, and screen six Sarawak rice landraces (Kanowit, Selasih, Merah, Merawi, Pandan Wangi and Bario) for resistance towards the newly isolated P. oryzae isolates. A total of 25 P. oryzae isolates were successfully isolated using spore-drop method and identified using conidia morphology and internal transcribed spacer (ITS) sequence. These 25 P. oryzae isolates were characterised for their morphological characteristics, growth rate and sporulation rate on two different media, oatmeal agar (OMA) and potato dextrose agar (PDA). The colony surface of all 25 P. oryzae isolates varied on different growing media whereas the pigmentation of upper colony surface varied from black, grey to white. There were no morphological traits that was unique to the division of the isolates originating from same location. The growth rate and sporulation rate for 24 P. oryzae isolates varies depending on the type of media used and differs among isolates. A total of 11 P. oryzae isolates from this study together with 15 Sarawak P. oryzae isolates from stock cultures of previous study and FRST UNIMAS Collection were used for AvrPiz-t, AvrPik and AvrPi-54 gene isolation. A total of 26 sequences of AvrPiz-t, 11 AvrPik and 17 AvrPi-54 genes from Sarawak isolates were successfully amplified and verified. The open reading frame (ORF) of the Avr genes were aligned with AvrPiz-t, AvrPik and AvrPi-54 ORF (respectively) from other countries that are available in National Centre for Biotechnology Information database. Unique sequences were identified and translated into amino sequence. The amino acid sequences were aligned with haplotype sequences from previous studies. In total, there were 19 AvrPiz-t haplotypes, with eight newly identified haplotypes, 12 AvrPik haplotypes with one newly identified haplotype and seven newly identified AvrPi-54 haplotypes. Neutrality tests were performed and suggested that AvrPiz-t and AvrPi-54 gene were under negative selection while AvrPik were under positive selection. Pathogenicity assay was performed on six selected Sarawak rice (Kanowit, Selasih, Merah, Merawi, Pandan Wangi and Bario) against five P. oryzae isolates from Sarawak (POS22, POS24, POSA20, POSA23 and POSI7) using detached-leaf inoculation. All rice landraces showed different levels of susceptibility towards all five P. oryzae isolates. Isolate POS22 was the most virulent isolate. Kanowit landrace was the most susceptible landrace and Merawi landrace was the least susceptible landrace. From alternative host, seven P. oryzae isolates from Eleusine indica were successfully obtained using the spore-drop method and confirmed through conidia morphology identification and ITS sequence. Morphological characterization revealed variations in upper colony surface on different growing media, with pigmentation ranging from black, grey, light grey to white. The growth and sporulation rates differed among the isolates and were influenced by the type of media used. The data collected is crucial for disease management in Malaysia, particularly in Sarawak, and serves as a reference for future rice breeding programs in the region.

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