Mekanisme ketahanan kakao terhadap penyakit busuk buah (Phytophthora palmivora)

Busuk buah (Phytophthora palmivora (Butl.) Butl.) merupakan penyakit kakao yang penting di banyak negara produsen tanaman ini, termasuk Indonesia. Walaupun aplikasi fungisida dapat mengurangi kerusakan akibat infeksi P. palmivora, cara ini mahal dan sering kali tidak dapat dilakukan oleh petani. Penggunaan bahan tanam yang tahan dianjurkan untuk mengatasi masalah ini. Informasi mengenai mekanisme ketahanan kakao terhadap penyakit busuk buah diperlukan dalam usaha pengendalian jangka panjang Mekanisme ketahanan kakao terhadap penyakit busuk buah dibedakan atas mekanisme struktural dan biokimia yang dapat bekerja sebelum penetrasi maupun setelah infeksi. Diduga ciri morfologi buah, seperti kedalaman dan lebar alur primer, kerapatan dan panjang mulut kulit merupakan salah satu mekanisme struktural yang berperan dalam ketahanan kakao terhadap penyakit busuk buah. Di samping itu, lignin asli maupun lignifikasi perikarp dapat menjadi mekanisme struktural yang membatasi serangan patogen setelah infeksi. Asam fenolat merupakan prekursor biosisntesis lignin. Asam ini berasal dari jalur fenilpropanoid. Jalur ini menghasilkan pula prekursor untuk sintesis tanin. Diduga kadar asam-asam ini meningkat setelah serangan patogen dan ini berhubungan dengan lignifikasi dan sintesis tanin. Bahan tanam yang diuji adalah Sca 12, TSH 858, DRC 16, ICS 60, NIC 4, dan GC 7. Uji ketahanan dilakukan pada buah yang dipetik dan di pohon, menggunakan metode titik (point inoculation) dengan kerapatan 106 spora kembara per ml. Penelitian mekanisme ketahanan meliputi pengamatan ciri morfologi buah, lignifikasi jaringan perikarp buah (menggunakan cat phloroglucinol-HCl dan lactophenol cotton blue). Macam senyawa fenolat yang dominan dan kandungannya dalam buah sebelum inokulasi, tiga dan enam hari setelah inokulasi, dianalisis dengan Kromatografi Cair Kinerja Tinggi (panjang gelombang 254 nm, kolom C 18). Hasil penelitian menunjukkan bahwa klon Sca 12 dan TSH 858 tahan, klon DRC 16 dan ICS 60 moderat, dan klon NIC 4 dan GC 7 rentan terhadap penyakit busuk buah. Uji pada buah yang dipetik dan di pohon menghasilkan aras ketahanan yang sama untuk masing-masing klon. Ada perbedaan ciri morfologi buah antarklon kakao. Kedalaman alur primer dan panjang pori mulut kulit berpengaruh besar pada ketahanan kakao terhadap penyakit busuk buah, dan kedua ciri morfologi ini menunjukkan hubungan erat yang bersifat negatif. Pengamatan irisan tipis menunjukkan bahwa lignin asli dalam perikarp buah kakao tidak dapat berperan dalam ketahanan sebelum penetrasi. Perubahan sitologi pascainfeksi pada klon tahan Sca 12 dan TSH 858 ditunjukkan dengan adanya granulasi sitoplasma sel-sel epidermis dan subepidermis dan terjadinya lignifikasi di sekitar sel-sel ini. Hasil analisis menunjukkan adanya asam klorogenat, asam ferulat, asam siringat, dan asam tanat dalam ekstrak perikarp buah kakao. Buah sehat (sebelum inokulasi) klon tahan Sca 12 dan TSH 858, dan klon moderat DRC 16 mempunyai kandungan senyawa ini yang lebih tinggi daripada klon moderat ICS 60, klon rentan NIC 4 dan GC 7, namun belum nyata peranannya dalam ketahanan sebelum penetrasi. Klon tahan mempunyai kandungan senyawa fenolat pascainfeksi yang lebih tinggi daripada klon moderat dan rentan. Infeksi P. palmivora menyebabkan peningkatan kandungan senyawa fenolat yang lebih awal pada klon tahan. Pada infeksi lanjut, kandungan asam klorogenat, asam ferulat, asam siringat, dan asam tanat klon rentan NIC 4 dan GC 7 lebih rendah daripada klon moderat dan tahan, sehingga kolonisasi patogen tidak terhambat. Di samping sebagai prekursor lignin, ekstrak senyawa fenolat total mampu menghambat pertumbuhan spora kembara P. palmivora in vitro. Ekstrak dari Sca 12 lebih mampu menghambat pertumbuhan patogen daripada klon lain. Toksisitas terbesar ditunjukkan oleh asam tanat. Kandungan asam klorogenat, asam ferulat, dan asam siringat mempunyai hubungan erat dengan kedalaman alur primer dan panjang pori mulut kulit. Ketahanan kakao terhadap penyakit busuk buah didasarkan pada mekanisme struktural (agregat sitoplasma dan lignifikasi ) dan mekanisme biokimia (kandungan asam fenolat dan asam tanat) yang bekerja pada tahap pascapenetrasi. Black pod caused by Phytophthora palmivora (Butl.) Butl. is the most important disease in cocoa producing countries, including Indonesia. Although chemical control method has been developed to reduce yield loss from P. palmivora infection, but the cost is still expensive and it often can not be used by farmers. The use of resistant planting material has been recommended as a more effective and economic control method. The understanding of disease resistance mechanism is needed in the long term control effort. The mechanism of cocoa resistance to P. palmivora has been distinguished to structural and biochemical mechanisms. These mechanisms are responsible to control resistance against pathogen penetration or postinfectional defense. Structural mechanism is attributed to pod morphological characteristics, such as width and depth of primary furrow, and density and pore length of stomata that influence cocoa black pod disease resistance. Both lignin and lignification of pod pericarp can serve as structural mechanisms, which can limiting the postinfectional growth of P. palmivora. Phenolic acids are precursors of lignin biosynthesis. These acids are synthesized through phenylpropanoid pathway. This pathway also yields some precursors for tannin synthesis. It was proposed that phenolic compounds will increase following pathogen attack and this is closely related to lignification and tannin synthesis. There are six clones of cocoa planting material used in this experiment, namely, Sca 12, TSH 858, DRC 16, ICS 60, NIC 4, and GC 7 clones. The resistance test was carried out on detached and attached pods, using point inoculation method with a zoospore suspension at a concentration of 106 ml-1. The resistance mechanism was observed, based on pod morphological characteristics and lignification of pod pericarp tissue (using phloroglucinol-HCl and lactophenol cotton blue staining). The type of dominant phenolic compound and their content in detached and attached pods before inoculation, three, and six days after inoculation was determined using High Performance Liquid Chromatography (at 254 nm wave length and C 18 column). The results showed that Sca 12 and TSH 858 were identified as resistant clones, while DRC 16, ICS 60, and NIC 4, GC 7 as moderate and susceptible clones, respectively. The level of resistance between detached and attached pod was similar for each clone. There were significantly difference in pod morphological characteristics among cocoa clones. Primary furrow depth and stomatal pore length seemed to be influence on cocoa resistance. It showed that there was a negatively close correlation between these characteristics. Native lignin in pod pericarp have not implicated to cocoa resistance to black pod pathogen. Histological change following pathogen infection was observed in resistant clone Sca 12 and TSH 858 that were indicated by the presence of granulated cytoplasm of epidermal and subepidermal cells, and lignification on cell around such granulated cell. HPLC analysis of healthy and diseased pod pericarp extract showed that phenolic compounds were identified as chlorogenic, ferulic, syringic, and tannic acids. Higher phenolic compound was measured in healthy pod of Sca 12, TSH 858, and DRC 16 than ICS 60, NIC 4, and GC 7. However, these compound seem not to be involved on the penetration resistence. Postinfection phenolic compound content of resistant clones was higher than that of moderate and susceptible clones. A rapid increasing of phenolic compounds after infection was detected on resistant clone. At six days after inoculation, lower phenolic acid content was observed in susceptible clone, NIC 4 and GC 7, than on moderate and resistant clones, therefore patogen colonization was not inhibited on these clones. It was demonstrated that phenolic compound extracted from cocoa pod was able to inhibit the growth of P. palmivora zoospores in vitro. The highest inhibition is resulted by Sca 12 extract. In this experiment, tannic acid was the most toxic compound to P. palmivora than the other phenolic compounds. It was also indicated that chlorogenic, ferulic, and syringic acid contents were correlated with primary furrow width and stomatal pore length. It was concluded that cocoa resistance to black pod pathogen is controlled by the structural mechanism which attributed to cytoplasmic aggregation and pod pericarp lignification; and by biochemical mechanism which involved at postinfection processes, such as the increase of phenolic compound contents