Sains Malaysiana 53(5)(2024): 1033-1042

http://doi.org/10.17576/jsm-2024-5305-05

Development of Real-time PCR Method for Detection of Xanthomonas campestris pv. juglandisCausing Walnut Bacterial Blight

(Pembangunan Kaedah PCR Masa Nyata untuk Pengesanan Xanthomonas campestris pv. juglandis Penyebab Hawar Bakteria Walnut)

YUQING DONG, LING XIE, HANMINGYUE ZHU & TIANHUI ZHU*

College of Forestry, Sichuan Agricultural University, Chengdu 611130, China

Diserahkan: 11 Julai2023/Diterima: 18 April 2024

Abstract

Walnut is one of the important economic tree species in the world. For the past few years, with the continuous expansion of walnut planting area, walnut bacterial blight caused by Xanthomonas campestrispv. juglandis has critically affected walnut production and caused economic losses. In this research, by comparing the gene sequences of X. campestrispv. juglandis and its closely related bacterial species, the 16S rDNA gene sequence was selected, and the specific primers were evaluated using PCR. Subsequently, the real-time fluorescence PCR specific detection method of X. campestrispv. juglandis was established and optimized. The specificity of real-time PCR was verified for 23 strains isolated from walnut and its surrounding soil, including eight target pathogens. The specific amplification was possible even when the cDNA template concentration was 7.0×10^-5 µg/mL, hence, the detection sensitivity of the standard method is 7.0×10^-5 µg/mL. The reliability of the established real-time PCR detection method in field detection was verified by the identification of target bacteria in 8 randomly collected healthy leaves and leaves of suspected walnut blight. This method laid a foundation for the early diagnosis of X. campestrispv. juglandis.

Keywords: Rapid detection; real-time fluorescence PCR; walnut bacterial blight; Xanthomonas campestrispv. juglandis

Abstrak

Walnut adalah salah satu spesies pokok yang penting dalam ekonomi dunia. Sejak beberapa tahun kebelakangan ini, dengan pengembangan berterusan kawasan penanaman walnut, hawar bakteria walnut yang disebabkan oleh Xanthomonas campestris pv. juglandis telah menjejaskan pengeluaran walnut secara kritikal dan menyebabkan kerugian ekonomi. Dalam penyelidikan ini, dengan membandingkan jujukan gen antara X. campestris pv. juglandis dan spesies bakteria yang berkait rapat, jujukan gen 16S rDNA telah dipilih dan pencetus khusus telah dinilai menggunakan PCR. Seterusnya, kaedah pengesanan khusus PCR pendarfluor masa nyata bagi X. campestris pv. juglandis telah dibangunkan dan dioptimumkan. Kekhususan PCR masa nyata telah disahkan untuk 23 strain yang dipencilkan daripada walnut dan tanah di sekelilingnya, termasuk 8 patogen sasaran. Amplifikasi khusus masih berlaku walaupun apabila kepekatan templat cDNA ialah 7.0×10-5 µg/mL, oleh itu, kepekaan pengesanan kaedah piawai ialah 7.0×10-5 µg/mL. Kebolehpercayaan kaedah pengesanan PCR masa nyata yang telah dibangunkan dalam pengesanan di lapangan telah disahkan dengan pengenalpastian bakteria sasaran dalam 8 daun sihat yang dikumpul secara rawak dan daun yang disyaki menghidapi hawar walnut. Kaedah ini meletakkan asas untuk diagnosis awal X. campestris pv. juglandis.

Kata kunci: PCR pendarfluor masa nyata; pengesanan yang pantas; penyakit hawar bakteria walnut; Xanthomonas campestrispv. juglandis

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*Pengarang untuk surat-menyurat; email:  zhuth1227@126.com