Isolasi, Karakterisasi, dan Uji Efektivitas Fungi Pendegradasi Pestisida Klorpirifos Asal Tanaman Bawang Merah (Allium Ascalonicum L.)

Abstract

Provinsi Jawa Tengah adalah wilayah dengan daerah pertanian terbesar kedua setelah provinsi Jawa Timur, dengan luas 966.647 ha. Luas lahan pertanian juga sebanding dengan produksi tanaman pangan dan hortikultura di Indonesia. Kabupaten Brebes yang terletak di Provinsi Jawa Tengah adalah sentra pusat produksi bawang merah. Bawang merah (Allium Ascalonicum L.) merupakan salah satu tanaman hortikultura yang bernilai ekonomi penting. Namun, kendala pada budidaya tanaman bawang merah yaitu tanaman ini memiliki kerentanan yang tinggi terhadap serangan hama dan penyakit tanaman. Oleh karena itu, petani bawang di Kecamatan Wanasari menggunakan sejumlah besar pestisida dan rata rata mengaplikasikan pestisida setiap tiga atau empat hari untuk mencegah kerusakan pada tanaman. Perairan tambak serta saluran irigasi di daerah Jawa Tengah telah tercemar oleh pestisida yang berasal dari limbah sektor pertanian. Penggunaan pestisida pasti meninggalkan residu pada tanah yang nantinya akan diserap oleh tanaman. Residu pestisida di lingkungan merupakan akibat buruk dari penggunaan atau aplikasi langsung pestisida. Pestisida yang ditujukan pada sasaran tertentu seperti tanaman dan tanah dapat terbawa oleh gerakan air, gerakan angin atau udara. Residu pestisida juga dapat berpotensi terbawa dalam rantai makanan. Degradasi residu bahan aktif pestisida serta perbaikan tanah tercemar pestisida dapat dipercepat dengan bantuan mikrob yang disebut dengan biodegradasi. Beberapa kelompok mikrob seperti bakteri dan fungi dapat mendegradasi bahan aktif organofosfat dalam tanah menjadi bentuk sederhana dan tidak beracun. Akumulasi residu pestisida yang semakin meningkat tentu dapat mengganggu keseimbangan lingkungan sehingga perlu dilakukan penelitian untuk mengurangi konsentrasinya di dalam tanah dengan pendekatan secara biologis agar tercipta food safety (keamanan pangan) serta kesehatan tanah dan lingkungan. Penelitian ini bertujuan untuk mengisolasi, skrining, identifikasi, dan mengkarakterisasi fungi pendegradasi pestisida klorpirifos dari lahan pertanian sentra produksi bawang merah, Kabupaten Brebes, Jawa Tengah dan Kabupaten Sumbawa, NTB serta untuk mengetahui mekanisme biodegradasi dan menguji aktivitasnya sebagai agen bioremediasi. Penelitian ini dilaksanakan pada bulan Mei 2021 sampai dengan bulan Desember 2021 di Laboratorium Biologi, Balai Penelitian Tanah, Jalan Tentara Pelajar No.12, Cimanggu, Bogor, Jawa Barat. Sampel tanah komposit diambil dari lahan pertanian bawang merah yang mempunyai riwayat penggunaan pestisida jenis klorpirifos dosis tinggi. Sampel tanah dan tanaman bawang merah diambil dari Kabupaten Brebes, Kecamatan Wanasari (6°52’40.8”S 109°00’18.0”E) dan Kabupaten Sumbawa, Kecamatan Moyo Hilir (8°34'18.8"S 117°29'06.1"E). Kegiatan penelitian terdiri dari: 1. Pengambilan sampel tanah terkontaminasi pestisida klorpirifos, 2. Isolasi, purifikasi, seleksi pada bahan aktif klorpirifos, profenofos, paraquat, dan glifosat, serta identifikasi morfologi dan molekuler isolat fungi pendegradasi klorpirifos, 3. Karakterisasi fungi, meliputi: uji kemampuan fungi dalam melarutkan fosfat dan kalium, uji patogenitas, uji kompatibilitas, uji kemampuan menghasilkan hormon tumbuh IAA, 4. Mekanisme biodegradasi dengan mengamati uji pemanfaatan klorpirifos sebagai sumber C dan P, dan 5. uji kemampuan fungi dalam mendegradasi pestisida secara in vitro. Sebanyak dua puluh sembilan isolat fungi berhasil diisolasi dari jaringan endofit dan sampel tanah tanaman bawang merah. Isolat WD1, WS1, dan WS2 mampu tumbuh pada klorpirifos 3000-6000 ppm, profenofos 4000-8000 ppm, paraquat 3000 ppm, dan glifosat 4000-8000 ppm. Isolat WS1 dan WS2 mempunyai aktivitas sebagai pelarut P dengan indeks pelarutan 0,23-0,29 dan secara kuantitatif mampu menghasilkan P sebanyak 4,83-5,02 ppm. Sementara itu, ketiga isolat fungi tidak mempunyai aktivitas dalam melarutkan K. Isolat fungi WD1, WS1, dan WS2 juga memiliki kemampuan menghasilkan IAA secara berturut-turut sebesar 0,148; 0,100; 0,103 ppm. Ketiga isolat aman digunakan karena tidak bersifat patogen pada tanaman dan hewan. Berdasarkan hasil analisis BLAST, isolat fungi WD1 diidentifikasi sebagai Fusarium falciforme dengan similaritas 99,82%. Hal ini didukung dengan analisis filogenetik yang menunjukkan isolat fungi tersebut berada dalam satu ranting dengan Neocosmospora falciformis dengan nilai boostrap sebesar 92%. Isolat WS1 dan WS2 diidentifikasi berdasarkan analisis BLAST sebagai Aspergillus foetidus dengan similaritas keduanya yaitu 99,66%. Analisis filogenetik menunjukkan bahwa kedua isolat tersebut berada dalam satu ranting dengan A. foetidus dengan nilai boostrap 97%. Fungi N. falciformis WD1 memiliki pertumbuhan yang lebih cepat, pada hari keenam memasuki fase eksponensial. Fungi A. foetidus WS1 mempunyai diameter paling besar pada media C, artinya fungi tersebut menggunakan klorpirifos sebagai sumber C lebih tinggi dari fungi lain. Fungi N. falciformis WD1 mempunyai diameter lebih besar dibandingkan fungi lain pada media P serta C dan P, sedangkan fungi A. foetidus WS2 paling rendah dalam menggunakan klorpirifos sebagai sumber energi yang ditandai dengan diameter yang rendah pada semua jenis media. Analisis biodegradasi in vitro menghasilkan fungi A. foetidus WS1 mampu mendegradasi klorpirifos (100 ppm) secara biotik dengan nilai penurunan tertinggi di antara perlakuan lainnya yaitu sebesar 9,33% dengan berat kering biomassa tertinggi yaitu 185 mg.

Central Java province is the second largest agricultural area after East Java province, with an area of 966,647 ha. The area of agricultural land is also comparable to the production of food crops and horticulture in Indonesia. Brebes Regency, located in Central Java Province, is the center of shallot production. Shallots (Allium Ascalonicum L.) is one of the horticultural crops that have important economic value. Shallots as one of the main commodities in the horticulture sub-sector after large chili and cayenne pepper commodities (Ministry of Agriculture 2015), has a high economic value (Santoso 2013). The economic value of shallots is higher than that of various other vegetables (Ngatindriatun et al. 2012). The production of shallots in Brebes Regency in 2014 reached 3,759,742 kw, the average production was 121.46 kw/ha. Wanasari district as the area with the highest onion production in Brebes is able to produce 1,025,680 kw, the average production is 144.97 kw/ha (BPS 2014). However, the problem with onion cultivation is that this plant has a high susceptibility to pests and plant diseases. According to the research of Joko et al. (2017) onion farmers in Wanasari sub-district use a large amount of pesticides and on average apply them every three or four days. Shallots are very susceptible to pests, so farmers use large amounts of pesticides to prevent damage to crops. Taufik et al. (2009) reported that pond waters and irrigation canals in Central Java have been polluted by pesticides originating from agricultural sector waste. Based on the survey results at the research site, one of the pesticides widely used by farmers around the waters of Mlonggo District, Jepara Regency, Central Java is organophosphate. The use of pesticides will leave residues on the soil that will be absorbed by plants. Pesticide residues in the environment are a bad result of the use or direct application of pesticides. Pesticides that are aimed at specific targets such as plants and soil can be carried away by the movement of water, wind or air movement. Pesticide residues can also potentially be carried along the food chain (Lucky 1991). The degradation of pesticide active ingredient residues and improvement of pesticide contaminated soil can be accelerated with the help of microorganisms called biodegradation. Several groups of microbes such as bacteria and fungi can degrade the active ingredients of organophosphates in the soil into simple and non-toxic forms. The increasing accumulation of pesticide residues can certainly disrupt the environmental balance, so research is needed to reduce its concentration in the soil with a biological approach in order to create food safety and soil and environmental health. This study aims to isolate, screening, and characterize the pesticide-degrading fungi chlorpyrifos from shallot production centers, Brebes Regency, Central Java and Sumbawa Regency, NTB as well as to determine the mechanism of biodegradation and test its activity as a bioremediation agent. This research will be carried out from May 2021 to December 2021 at the Biology Laboratory, Soil Research Institute, Jalan Tentara Pelajar No. 12, Cimanggu, Bogor, West Java. Composite soil samples were taken from shallot farms that have a history of using high doses of organophosphate pesticides. Soil and shallot plant samples were taken from Brebes Regency, Wanasari District (6°52'40.8”S 109°00'18.0”E) and Sumbawa Regency, Moyo Hilir District (8°34'18.8"S 117°29'06.1" E). The research treatments consisted of: 1. Sampling of soil contaminated with the organophosphate pesticide type chlorpyrifos, 2. Isolation, purification, selection on four different active ingredients (chlorpyrifos, profenofos, paraquat, and glyphosate) and identification of morphology and microscopy of isolates of chlorpyrifos-degrading fungi, 3 Characterization of fungi, including: test the ability of fungi to dissolve phosphate and potassium, pathogenicity test, compatibility test, test the ability to produce growth hormone IAA, 4. Biodegradation mechanism which includes: growth curve analysis, test the utilization of organophosphates as a source of C and P, and test ability of fungi to degrade pesticides in vitro, 5. Identification and molecular characterization of fungi. The results obtained on the isolation of fungi from endophytic tissue and soil samples of shallot plants as many as twenty-nine isolates of fungi which were then screened. The results of the screening showed that three isolates were able to grow at the highest concentration of chlorpyrifos and other active ingredients, namely isolates WD1, WS1, and WS2. The fungi were tolerant to 3000-6000 ppm chlorpyrifos, 4000-8000 ppm profenofos, 3000 ppm paraquat, and 4000-8000 ppm glyphosate. The isolates WS1 and WS2 had activity as P solvents with a dissolution index of 0.23-0.29 and quantitatively were able to produce P as much as 4.83-5.02 ppm. Meanwhile, the three fungal isolates did not have activity in dissolving K. Fungal isolates WD1, WS1, and WS2 also had the ability to produce IAA of 0.148, respectively; 0.100; 0.103 ppm. The three isolates were safe to use because they were not pathogenic isolates in plants, animals, and humans according to hypersensitivity and hemolysis tests. Based on the results of BLAST analysis, WD1 isolate was identified as Fusarium falciforme with 99.82% similarity. This was supported by a phylogenetic analysis which showed that the fungal isolates were in the same branch as Neocosmospora falciformis with a boostrap value of 92%. Isolates WS1 and WS2 were identified based on BLAST analysis as Aspergillus foetidus with a similarity of 99.66%. Phylogenetic analysis showed that the two isolates were in the same branch with A. foetidus with a boostrap value of 97%. The biodegradation ability test was obtained from the results of the growth curve of the fungus N. falciformis WD1 which had a faster growth, on the sixth day it entered the exponential phase. The results of the use of C and P showed that A. foetidus WS1 had the largest diameter on C media, meaning that the fungus that used chlorpyrifos as a source of C was higher than other fungi. The fungus N. falciformis WD1 had a larger diameter than other fungi on P and C and P media, while A. foetidus WS2 was the lowest in using chlorpyrifos as an energy source, which was characterized by a low diameter in all types of media. In vitro biodegradation analysis resulted that the fungus A. foetidus WS1 was able to degrade chlorpyrifos (100 ppm) biotically with the highest reduction value among other treatments, namely 9.33% with the highest dry weight biomass in a single isolate, namely 185 mg.