Genetic diversity and microevolution of pyricularia grisea from grasses

Abstract

Pyricularia is the fungus caused rice and grasses blast disease. They are morphologically indistinguishable, therefore their specific epithet is often based on host. The concept of Pyricularia species using nucleotide sequences of current molecular approach is still on controversy, especially those infected weed grasses (Pyricularia grisea) and rice (Pyricularia oryzae). Yet genetic change caused by host alteration has not been explored. Therefore, this researchs were aimed to (i) study the populations diversity of Pyricularia from various grasses (Cynodon dactylon, Digitaria ciliaris, Eleusine indica, and Panicum repens) using five markers that consist of three kinds of sequence characterized amplified region (SCAR), i.e Cut1, PWL2 and Erg2 and two markers, magB and magC, repectively; (ii) to analyze genetic diversity of the basal samples of Pyricularia from Digitaria ciliaris and its microevolution of Pyricularia d4 from D. ciliaris following cross infection to rice and Panicum repens by adding markers of amplified fragment length polymorphism (AFLP), PCR of repetitive Pot2 (rep- Pot2), pathotype, and sequences of ITS and 5.8S rDNA nucleus; (iii) study phylogenetic relationship of Pyricularia all of from D. ciliaris (d4), P. repens (pr10.a.S4), and from rice pathogen (ok6, ou6.S4) based sequences of ITS and 5.8S rDNA. This study used Pyricularia from grasses and rice from West Java that occurs in the same field as a model. The result showed DNA size of all markers of Cut1, PWL2 and Erg2, magB, and magC of Pyricularia from grasses were similar to that of rice origin. The frequency of those SCAR markers, i.e. Cut1, PWL2, and Erg2 were 78.1%, 54.1% and 95.1%, respectively. Based on present SCAR markers, they could be classified into five phenotypes (011, 101, 111, 001, 010) out of the eight possible groups. Cross infection of Pyricularia d4 from D. ciliaris grass to rice could induce genetic variation in their Cut1 and PWL2 markers, AFLP and rep-Pot2 phenotypes, as well as pathotype. On the other hand, no new variation revealed their ITS sequences. Moreover, the cross infection to another grass in different genus (P. repens) also caused genetic variation in AFLP and rep-Pot2 phenotypes, and ITS sequences. These results indicated that the cross infection might induce microevolution of Pyricularia d4. Sequences of ITS and 5.8S rDNA of Pyricularia all of from D. ciliaris (d4), and from rice pathogen (ok6, ou6.S4) were similar. Whereas those of Pyricularia from P. repens (pr10.a.S4) had only one nucleotide different in their 5.8S rDNA sequences to those of Pyricularia d4. BLAST analysis of ITS and 5.8S rDNA sequences from five isolates showed 99% identical to Magnaporthe oryzae pathogen of rice, cereals and cultivated grasses. The ITS sequences of the non-rice isolates were similar with isolates of the rice blast disease, so there might be gene flow among the pathogen on grasses and rice. Based on nomenclatural priority, the specific name for Pyricularia from D. ciliaris, P. repens, and rice were Pyricularia grisea as the anamorphic of Magnaporthe grisea.

Rumput yang tumbuh di sekitar pertanaman padi telah dilaporkan sebagai inang Pyricularia. Pyricularia penginfeksi rumput memiliki keragaman genetik berbeda terhadap Pyricularia yang menginfeksi padi. Sampai saat ini analisis keragaman genetik Pyricularia dari rumput yang mengalami pergantian inang pada genus berbeda belum pernah dilaporkan. Pergantian inang atau perubahan patogenisitas diduga dapat menjadi faktor pendorong mikroevolusi pada Pyricularia. Mikroevolusi merupakan perubahan yang dapat tampak dalam waktu relatif pendek (beberapa hari atau minggu) pada mikrob. Mikroevolusi Pyricularia yang mengalami pergantian genus inang juga belum pernah diteliti. Spesies epitet grisea dari rumput dan oryzae dari padi ditetapkan berdasarkan inang. Kedua spesies tidak berbeda morfologinya. Berkembangnya berbagai pendekatan spesies, menyebabkan konsep spesies Pyricularia belum disetujui bersama. Couch dan Kohn pada tahun 2002 menempatkan Pyricularia yang patogen pada Digitaria sanguinalis sebagai Pyricularia grisea, yaitu sama seperti prinsip penamaan sebelumnya. Isolat-isolat Pyricularia dari rumput dan padi yang diperoleh dari ladang dan sawah di Jawa Barat menjadi model pada penelitian ini. Hasil penelitian ini diharapkan dapat berkontribusi dalam bentuk informasi pada manajemen penyakit blas pada padi di Jawa Barat. Penelitian ini bertujuan menganalisis (i) keragaman populasi Pyricularia dari rumput (Cydonon dactylon, Eleusine indica, Digitaria ciliaris, dan Panicum repens) berdasarkan lima penanda yang terdiri atas tiga penanda (Cut1, PWL2 dan Erg2) sequence characterized ampllified region (SCAR), dan dua penanda berupa magB, dan magC, (ii) keragaman genetik sampel basal Pyricularia dari rumput Digitaria ciliaris dan mikroevolusi Pyricularia d4 yang berasal dari Digitaria ciliaris sebagai hasil induksi pergantian inang ke padi dan ke rumput Panicum repens berdasarkan lima penanda, yaitu SCAR, amplified fragment length polymorphism (AFLP), PCR pada repetitive Pot2 (rep-Pot2), dan hubungannya dengan ras fisiologi, serta sekuen ITS beserta 5.8S rDNA nukleus yang dihasilkan melalui amplifikasi dengan primer universal berupa its1 dan its4; (iii) hubungan filogenetik Pyricularia dari rumput dan padi berdasarkan sekuen ITS beserta 5.8S rDNA nukleus. Sekuen tersebut juga dihasilkan melalui amplifikasi dengan primer universal (its1 dan its4). Penelitian ini diharapkan dapat mengungkapkan peran pergantian inang pada perubahan genetik yang merupakan bagian dari mikroevolusi Pyricularia, dan hubungan filogenetik antara Pyricularia yang menginfeksi rumput dan padi.