Potensi Bakteri Selulolitik dan Asap Cair Ampas Bromelain untuk Pengendalian Busuk Hati (Phytophthora nicotianae) Nanas

Abstract

Penyakit busuk hati pada tanaman nanas yang disebabkan oleh Phytophthora nicotianae merupakan salah satu kendala utama dalam produksi nanas karena bersifat tular tanah dan sulit dikendalikan. Patogen ini termasuk kelompok Oomycota yang memiliki dinding sel mengandung selulosa, sehingga berpotensi dikendalikan menggunakan agen hayati penghasil enzim selulase. Selain itu, pemanfaatan limbah agroindustri seperti ampas bromelain nanas juga berpotensi dikembangkan sebagai fungisida nabati yang ramah lingkungan. Penelitian ini bertujuan untuk mendapatkan isolat bakteri selulolitik yang potensial dalam mengendalikan penyakit busuk hati oleh P. nicotianae pada tanaman nanas serta memperoleh informasi mengenai keefektifan asap cair ampas bromelain dalam menekan pertumbuhan P. nicotianae. Penelitian mengenai potensi bakteri selulolitik diawali dengan isolasi rhizobakteri dari rizosfer tanaman famili Bromeliacea, diikuti dengan identifikasi patogen P. nicotianae secara morfologi dan molekuler. Isolat bakteri diseleksi berdasarkan kemampuan selulolitiknya menggunakan media carboxymethyl cellulose (CMC) dan diukur indeks selulolitiknya. Isolat terpilih selanjutnya diuji keamanan hayati dan daya antagonisnya terhadap P. nicotianae melalui uji kultur ganda, uji senyawa volatil secara in vitro, serta uji semi in vivo. Pemilihan isolat potensial dilakukan menggunakan metode analytic hierarchy process (AHP). Isolat terpilih kemudian dikarakterisasi secara fisiologis, meliputi kemampuan menghasilkan hormon indole-3-acetic acid (IAA) dan melarutkan fosfat, serta diidentifikasi secara molekuler. Hasil identifikasi secara morfologi pada media CMA dan media ISP-2, diketahui P. nicotianae memiliki hifa bercabang dan tidak bersekat, berwana hyaline, serta menghasilkan sporangium reproduksi aseksual dan oospora reproduksi seksual. Fragmen pita hasil amplifikasi yang tervisualisasi pada ukuran 900 bp dengan primer ITS1 dan ITS4 dengan nilai query cover sebesar 100% dan tingkat kesamaan sekuens (homology) sebesar 100% terhadap seluruh sekuens pembanding, memiliki kekerabatan dengan P. nicotianae dengan beberapa strain, strain CTA3K1a2, BG4K3b, BY1Q1g, ZYMQ1a, dengan berbagai negara serta tanaman inang yang berbeda. Kekerabatan isolat patogen berdasarkan nilai boostrap tertinggi, berada pada P. nicotianae strain CTA3K1a2 pada tanaman citrus di Tunisia. Sebanyak 19 isolat bakteri selulolitik yang ditemukan sebagai kandidat agens hayati yang aman secara hayati dan memiliki kemampuan selulolitik yang bervariasi. Uji kultur ganda menunjukkan bahwa isolat A45 dan FTA22 memiliki daya hambat tertinggi terhadap P. nicotianae, masing-masing sebesar 47,11% dan 40,99%. Pada uji senyawa volatil, isolat FT57 dan K5 menunjukkan penghambatan terbesar terhadap pertumbuhan P. nicotianae, masing-masing sebesar 55,18% dan 44,98%. Uji semi in vivo terhadap lima isolat terpilih hasil seleksi AHP menunjukkan bahwa seluruh isolat mampu menghambat perkembangan lesi penyakit busuk hati, dengan persentase penghambatan tertinggi adalah isolat FT57 dan A45, masing-masing sebesar 73,74% dan 71,81%. Kedua isolat tersebut juga mampu menghasilkan hormon IAA, melarutkan fosfat, serta memiliki indeks selulolitik masing-masing sebesar 1,83 dan 1,18. Identifikasi secara molekuler menggunakan primer 27F dan 1492R diperoleh bahwa isolat FT57 berkerabat dekat dengan Bacillus subtilis MS7, sedangkan isolat A45 memiliki kemiripan dengan Stenotrophomonas maltophilia strain JFZ2. Potensi asap cair ampas bromelain dilakukan melalui penyiapan larutan asap cair dan kultur P. nicotianae, kemudian diuji efikasinya menggunakan metode peracunan media pada berbagai konsentrasi. Pertumbuhan koloni P. nicotianae diamati selama periode inkubasi, dan nilai konsentrasi hambat 50% (EC50) dan 90% (EC90) dihitung saat hari terakhir pengamatan kontrol memenuhi permukaan media. Hasil penelitian menunjukkan bahwa nilai EC50 asap cair ampas bromelain terhadap P. nicotianae sebesar 0,0448 mL mL-1 pada hari terakhir, sementaranilai EC90 sebesar 0,3626 mL mL-1. Secara keseluruhan, hasil penelitian ini menunjukkan bahwa bakteri selulolitik, khususnya isolat FT57 dan A45, serta asap cair ampas bromelain nanas memiliki potensi yang besar untuk dikembangkan sebagai alternatif pengendalian hayati penyakit busuk hati pada tanaman nanas yang ramah lingkungan dan berkelanjutan.

Heart rot disease of pineapple caused by Phytophthora nicotianae is one of the major constraints in pineapple production due to its soil-borne nature and difficulty to control. This pathogen belongs to the Oomycota group, which possesses cellulose-containing cell walls, indicating its potential susceptibility to biological control agents producing cellulolytic enzymes. In addition, the utilization of agro-industrial waste such as pineapple bromelain residue has the potential to be developed as an environmentally friendly botanical fungicide. This study aimed to obtain potential cellulolytic bacterial isolates for controlling heart rot disease caused by P. nicotianae in pineapple and to evaluate the effectiveness of liquid smoke derived from bromelain residue in suppressing the growth of P. nicotianae. The study on cellulolytic bacteria began with the isolation of rhizobacteria from the rhizosphere of plants belonging to the Bromeliaceae family, followed by morphological and molecular identification of P. nicotianae. Bacterial isolates were screened based on their cellulolytic activity using carboxymethyl cellulose (CMC) medium, and the cellulolytic index was determined. Selected isolates were subsequently evaluated for biosafety and antagonistic activity against P. nicotianae through dual culture assays, in vitro volatile compound assays, and semi in vivo tests. The selection of potential isolates was performed using the analytic hierarchy process (AHP). The selected isolates were further characterized physiologically based on their ability to produce indole-3-acetic acid (IAA) and solubilize phosphate, and were identified molecularly. Morphological identification on CMA and ISP-2 media showed that P. nicotianae possessed branched, aseptate, hyaline hyphae and produced sporangia as asexual reproductive structures and oospores as sexual reproductive structures. PCR amplification using ITS1 and ITS4 primers yielded a fragment of approximately 900 bp with 100% query coverage and 100% sequence homology to reference sequences of P. nicotianae, showing close relationships with several strains, including CTA3K1a2, BG4K3b, BY1Q1g, and ZYMQ1a from different countries and host plants. Phylogenetic analysis based on the highest bootstrap value indicated that the pathogen isolate was most closely related to P. nicotianae strain CTA3K1a2 from citrus in Tunisia. A total of 19 cellulolytic bacterial isolates were identified as candidate biological control agents that were biosafe and exhibited varying levels of cellulolytic activity. Dual culture assays revealed that isolates A45 and FTA22 showed the highest inhibition against P. nicotianae, with inhibition rates of 47.11% and 40.99%, respectively. In the volatile compound assay, isolates FT57 and K5 exhibited the greatest inhibition of P. nicotianae growth, with inhibition values of 55.18% and 44.98%, respectively. Semi in vivo tests on five selected isolates based on AHP showed that all isolates were able to suppress the development of heart rot lesions, with the highest inhibition observed in isolates FT57 and A45 at 73.74% and 71.81%, respectively. Both isolates were capable of producing IAA, solubilizing phosphate, and exhibited cellulolytic indices of 1.83 and 1.18, respectively. Molecular identification using primers 27F and 1492R revealed that isolate FT57 was closely related to Bacillus subtilis MS7, while isolate A45 showed high similarity to Stenotrophomonas maltophilia strain JFZ2. The potential of liquid smoke derived from bromelain residue was evaluated through the preparation of liquid smoke solutions and P. nicotianae cultures, followed by efficacy testing using the poisoned food technique at various concentrations. Colony growth of P. nicotianae was observed throughout the incubation period, and inhibitory concentration values of 50% (EC50) and 90% (EC90) were calculated at the final observation day when the control treatment completely covered the surface of the growth medium. The results showed that the EC50 value of liquid smoke from bromelain residue against P. nicotianae was 0.0448 mL mL?¹ on day 13, whereas the EC90 value was 0,3626 mL mL?¹. Overall, the results of this study indicate that cellulolytic bacteria, particularly isolates FT57 and A45, as well as liquid smoke derived from pineapple bromelain residue, have strong potential to be developed as environmentally friendly and sustainable alternatives for the biological control of heart rot disease in pineapple.