臺灣稻熱病菌AVR-Pik基因型組成與致病性之分析

Abstract

稻熱病（rice blast disease），為子囊真菌Magnaporthe oryzae所造成的水稻重要病害，其可感染水稻葉、莖及穗，每年皆造成全球水稻產量嚴重的損失，對糧食安全具有重大威脅。稻熱病重要的防治策略之一，為種植抗病品種，但由於田間稻熱病菌具有高度的遺傳變異性，使得原本抗病的水稻品種在推出幾年後，便逐漸失去抗病性，其中最顯著的例子為稻熱病菌avirulence（AVR） gene之變異。本研究探討臺灣稻熱病菌AVR-Pik基因型之組成及其遺傳變異，首先自IRRI LTH IRBL判別品系病圃及臺灣各地田間，採集病葉並利用單孢分離法分離稻熱病菌株，進行核酸之萃取。接著，以聚合酶連鎖反應（PCR）增幅AVR-Pik基因，經由選殖及DNA定序發現，臺灣稻熱病菌AVR-Pik之基因型主要為A及D兩類型，而A型中發現分別有兩種跳躍子（transposable element）之插入性突變，其一為Mg-SINE插入於AVR-Pik基因之ORF區域（AI），而另一為Pot3插入AVR-Pik基因之終止序列（terminator）區域（AII）；利用變異性菌株進行病原性測定（pathogenicity assay），顯示此兩種插入性突變皆會導致AVR-Pik A型基因功能的喪失。進一步選定2016至2018年間三個不同監測病圃，進行族群AVR-Pik基因型組成之分析，發現三個地區稻熱病菌族群AVR-Pik基因型主要皆為A+D之混合型菌株，而第二大主要族群於臺東關山病圃為AI+D之混合型，而屏東長治病圃及新竹峨嵋病圃兩處，則皆為A+AI+D之混合型。此外，選定臺灣商業化品種台南11號、台稉糯3號、台農77號及台農79號，進行水稻稻熱病Pik抗性基因之分析，結果顯示台南11號與文獻中的Piks胺基酸序列完全一致，而台稉糯3號、台農77號及台農79號則與Pik序列最為相近，僅在Pik1第465胺基酸位點由絲胺酸（Serine）轉變為脯胺酸（Proline）。田間種植具有垂直抗性基因的水稻抗病品種會增加稻熱病菌生存的選汰壓力，導致病原菌相對應的AVR基因變異喪失功能，進而造成田間抗性的消失，因此持續監控稻熱病菌株AVR基因的動態變異情形，將有助於病害流行的預防，以及抗病品種的選育與種植推廣策略。

Rice blast disease is caused by the ascomycete Magnaporthe oryzae, which can infect rice leaves, stems and spikes. The disease leads to severe economic loss around the world every year. To control rice blast disease, breeding and deployment of resistant varieties is one of the effective methods in many countries. However, due to highly genetic variation of rice blast fungi, breakdown of resistant varieties often occurs within few years when released for commercial production in monoculture fields. The most significant example is the mutation of M. oryzae avirulence (AVR) gene. The objective of this study is to determine the composition of AVR-Pik genotype among blast population by surveying IRRI LTH IRBL disease monitoring nursery and commercial fields in Taiwan. Firstly, blast fungi were isolated by single conidial isolation from leaf blast samples. M. oryzae DNA was extracted and then subjected to AVR-Pik gene PCR identification. According to DNA cloning and sequencing results, we found AVR-Pik A and D allele types in the blast population of Taiwan. Interestingly, two types of transposon insertional mutation were identified in AVR-Pik A allele among Taiwan population, including Mg-SINE inserted at ORF (AI) and Pot3 inserted at the terminator region (AII). According to pathogenicity assay, both mutations resulted in the loss of AVR-Pik-A function. We further selected three different IRRI LTH IRBL disease monitoring plots and analyzed AVR-Pik genotype composition among blast population from 2016 to 2018. The results showed that AVR-Pik A+D was the major genotype. The second largest population, AI+D, was mainly discovered in Guanshan, Taitung County. However, the second largest population found in Changzhi, Pingtung County and Ermei, Hsinchu County was A+AI+D genotype. Furthermore, a number of commercial rice varieties including TN11, TKW3, TNG77 and TNG79 were selected to analyze their Pik blast resistant gene. The results revealed that TN11 Pik1 gene was identical to Piks, whereas Pik1 alleles of TKW3, TNG77 and TNG79 were most similar to Pik allele except amino acid substitution at 465 position from Serine to Proline. The resistant rice varieties carrying vertical resistance gene pose survival pressure to blast population in the fields. The blast fungi then evolve to lose the function of AVR gene and consequently break down the field resistance. Therefore, pathogen surveillance such as monitoring the occurrence and dynamics of mutated AVR alleles will help prevent the epidemics of blast disease and set the strategies for breeding and deployment of resistant rice varieties for commercial production.