Ketahanan kacang tanah dan tembakau terhadap infeksi Sclerotium rolfsii Sacco dengan ekspresi enzim kitinase tinggi

Abstract

Peanut stem rot disease is caused by fungus Sclerotium rolftii Sacco The infested field soil by the fungus is difficult to control because the fungus can produce sclerotia, survive on death celVtissue, and omnipathogenic. Understanding of the plant resistance mechanism to control the patogen is required. Since the cell wall contain high chitin, an opportunity to enhance the peanut resistailce to S. rolftii infection by the increasing of chitinase activity. Characteristic of chitinase activity on peanut was observed to know the role of the enzyme to resistance mechanism to S. rolfsii. Both endo- and exo-chitinase activities were detected on all tested genotype. The resistant genotype showed high endo-chitinase activity. Under the standardization method, the chromogenic dimer was the most suitable substrate for analysing chitinase activity on peanut. The chitinase activity and response of the stemlbranchtissue were resemble to the root neck tissue (as an initial infection of S. rolfsii) but not the same as that of the leaf tissue. Therefore, the branch tissue can be used to analyze enzyme activity and to evaluate response to S. rolftii infection, to avoid destruction of the evaluated plant. The use of the root neck tissue for evaluation or analysis cause the susceptible plant die and further lost opportunity for the zuriat evaluation, which is sometime needed in plant breeding. Furthermore, the standardized methods were used to evaluate chitinase activity for 24 peanut genotypes which were spread allover Indonesia, consist of 11 cultivars and 13 local lines. Other physiological characters from the 24 genotypes as a peanut resistance response to S. rolfsii were also observed. Peroxidase activity has also an important role in the peanut resistance mechanism. The rapid formation reaction of oxidative-burst did not induced by S. rolftii infection. On the other hand, infection of the pathogen resulted in increasing lignin content in the infected tissues but not related to disease severity. This indicated that S. rolfsii was able to degrade lignin as that of a part of other species of Basidiomycetes fungi. !!l vitro selection methods could be used to improve the resistance to biotic and abiotic factors. The success of somaclonal induction application to improve single character, provide some researchers to improve more than one plant character simultaneously. Regeneration of varian somaclonal population via in vitro selection using S. rolfsii culture filtrate and PEG as a drought stress simultaneously has been done. In this case, enhance the resistance was determined by the material of fungi culture filtrat, namely toxin and element comprised the fungi micelial cell wall. Roles of chitinase activity in resistance mechanism to S. rolftii could be studied on peanut derived from this in vitro selection. The role of chitinase activity in the resistance mechanism as indicated on the peanut germplasm and in vitro selection production, and the availability of chi gene provide opportunity to the regeneration of S. rolftii resistant lines through genetic engineering to obtain over-expression chitinase activity transgenic peanut. Preffered genetic transformation into the model plant (tobacco), regeneration of the transgenic tobacco, and transgene expression detection were the easy and quick methodes to evaluate the effectiveness of genetic transformation. Percentage of the succeed genetic transformation of tobacco cv. Gombel Sili by the chi-rice gene was 0.1 %. Induced chitinase activity in the transformans T o-chi infected tissue were enhanced 4-14 fold compare to the activity on non-transformated tobacco. The inserted chi gene did not influence the peroxidase activity .. Evaluation of tobacco transgenic chi generation T1 showed that integration of chi transgene into tobacco genome could increase the chitinase activity. The increase of the chitinase activity was related to the enhanced resistance to S. rolfsii infection, both to SKc and SKps isolates, but did not enhance resistance to Phytophthora capsici. Resistance response of tobacco transgenic chi was determined based on the disease severity (Resistant-OS) and the sensitivity index (Resistant-SI). Apparently, this was due to the main composition of P. capsici mycelia cell wall was polymer ~- 1,3-glucan and cellulose contained low chitin, not as other fungi cell wall. Percentage of the succeed genetic transformation of peanut using cv. Singa is higher than cv. Kelinci. The genetic transformation in this experiment invented three putative peanut transgenic plants of cv. Kelinci and 13 plants of cv. Singa. The putative transgenic chi cv. Singa expressed chitinase activity, resistance response to S. rolfsii infection, and the coefficient determination of regression was higher than cv. Kelinci.

Penyakit busuk batang pada kacang tanah disebabkan oleh cendawan Sclerotium rolfsii Sacco Pada lahan-Iahan yang sudah terinfestasi cendawan ini, sulit untuk mengendalikannya karena cendawan mampu membentuk sklerotia, mampu bertahan hidup pada jaringan mati (nekrotropik), dan bersifat omnipathogenic. Pemahaman tentang mekanisme ketahanan tanaman terhadap infeksi patogen diperlukan untuk pengendalian. Dinding sel rniselia cendawan ini mengandung kitin tinggi sehingga membuka peluang upaya peningkatan ketahanan tanaman kacang tanah terhadap infeksi S. rolfsii dengan meningkatkan aktivitas kitinase. Karakteristik aktivitas kitinase pada tanaman kacang tanah dipelajari untuk mengetahui peran enzim ini pada mekanisme ketahanan terhadap S. rolfsii. Aktivitas endo-kitinase dan ekso-kitinase diamati pada semua genotipe yang diuji, genotipe tahan menunjukkan aktivitas endo-kitinase yang lebih tinggi. Pada pembakuan metode, penggunaan substrat dimer paling cocok untuk menganalisis aktivitas kitinase pada tanaman kacang tanah. Aktivitas kitinase dan respons jaringan batang terhadap infeksi S. rolfsii lebih mirip dengan jaringan leher akar (sebagai tempat awal infeksi S. rolfsii) dibandingkan dengan jaringan daun. Oleh karena itu, jaringan batang dapat digunakan untuk mengevaluasi respons dan aktivitas enzim yang berkaitan dengan infeksi cendawan ini, untuk menghindari evaluasi yang bersifat destruktif. Penggunaan jaringan leher akar untuk evaluasi dapat menyebabkan kematian pada individu yang rentan sehingga menghilangkan kesempatan untuk mengevaluasi zuriat dari tlinaman rentan yang seringkali diperlukan dalam bidang pemuliaan tanaman.