2. 580 Selection (Dwii Sugipr... Selection of Yeasts Antagonists as Biocontrol Agent of Mango Fruit Rot caused by Botryodiplodia theobromae DWI SUGIPRIHATINI , SURYO WIYONO , WIDODO 1 2* 2 AND 1 2 Balai Besar Uji Standar Karantina Pertanian, Jalan Pemuda No 64 Kav16-17, Rawamangun, Jakarta 13220, Indonesia Department of Plant Protection, Faculty of Agriculture, Institut Pertanian Bogor, Darmaga Campus, Bogor 16680, Indonesia Botryodiplodia theobromae B. theobromae Cryptococcus albidus aerius Pichia guilliermondii Debaryomyces hansenii B. theobromae C. albidus aerius C. albidus C. terreus Candida edax Candida edax Cryptococcus luteolus Botryodiplodia theobromae Cryptococcus albidus Botryodiplodia theobromae B. theobromae Cryptococcus albidus aerius Pichia guilliermondii Debaryomyces hansenii B. theobromae C. albidus aerius C. albidus C. albidus C. terreus Candida edax Candida edax C. luteolus Cryptococcus albidus Botryodiplodia theobromae ; Fruit rot caused by is one of the most important post harvest disease of mango in Indonesia. Study on biological control on the disease is required to develop environmentally-sound control technology. The research objectives were to study the potency of yeasts in controlling post harvest mango disease i.e. fruit rot caused by and mechanism involve in the biocontrol. Total yeast isolates used for screening were twenty one, four from collection of Plant Clinic of Institut Pertanian Bogor, and twenty one isolated from healthy mango skin. All of yeast isolates were characterized and identified using BIOLOG. Bioassay on antagonistic activity of yeasts against fruit rot, in-vitro dual culture test and chitinolytic activity were carried out. var. WSW1, K1, and K12 were the three most effective antagonistic yeasts against with effectiveness of 70.83, 45.83, 37.50% respectively. In vitro bio-assay showed that var. WSW1, WSW2, C. albidus K6, YSW1, OSW1, K13, and K2 had high antibiosis activity. Biocontrol activity of tested yeasts against fruit rot of mango did not correlate to its antibiosis and chitinolytic activity. Key words: biocontrol, , , mango, yeast Busuk buah yang disebabkan merupakan salah satu penyakit pasca panen mangga terpenting di Indonesia. Penelitian tentang pengendalian hayati penyakit tersebut diperlukan untuk mengembangkan teknologi pengendalian yang ramah lingkungan. Penelitian ini bertujuan mengkaji potensi khamir antagonis dalam pengendalian busuk buah mangga yang disebabkan dan beberapa mekanisme yang terlibat. Isolat khamir diperoleh dari koleksi Klinik Tanaman Institut Pertanian Bogor (IPB), yang diisolasi oleh salah seorang penulis dan diisolasi dari buah mangga sehat. Isolasi khamir dilakukan dengan pencucian kulit buah yang dilanjutkan dengan penanaman pada media PDA pH 5.5. Jumlah isolat khamir yang diseleksi semuanya ada 21, 4 dari koleksi Klinik Tanaman IPB dan 17 dari hasil isolasi penulis. Semua isolat khamir identifikasi dengan BIOLOG. Semua isolat khamir dikaji kemampuan antagonisnya terhadap busuk buah, uji koloni ganda in-vitro dan uji aktivitas kitinase. var. WSW1, K1, dan K12 adalah tiga isolat khamir antagonis paling efektif terhadap dengan tingkat penekanan secara berturut-turut 70.83, 45.83, dan 37.50%. Uji koloni ganda in-vitro menunjukkan bahwa var. WSW1, WSW2, K6, YSW1, OSW1, K13, dan K2 mempunyai aktivitas antibiosis yang tinggi. Keefektifan pengendalian hayati khamir yang diuji terhadap busuk buah mangga tidak berkorelasi dengan aktivitas antibiosis dan kitinase khamir tersebut. Kata kunci: , , khamir, mangga, pengendalian hayati Mango is one of important tropical fruit commodity for many tropical countries for domestic trade and -also export. One major post harvest disease in Indonesia is fruit rot caused by , causing serious damage in storage and shipping. Infection rate of disease on mango fruit Arumanis was average 54% (Yulianingsih . 1990). There are no effective control measures against this disease. Existing control measures is the application of fungicide after harvest. The fungicide use has low consumer's acceptance due to environment and health issues. Biological control using yeasts is a promising alternative to control post harvest diseases of fruits and Botryodiplodia theobromae cv. et al vegetables. The advantages using yeast for biocontrol agent it grows fast, dry tolerance; do not produce mycotoxin and allergenic spores (Droby and Chalutz 1994). Previous research showed that yeasts can be applied to delay fruit ripening process and disease control as well (Janisiewicz and Korsten 2002). Delay of fruit ripening by yeast is through inhibition of ethylene production (Droby 1997). Some research showed that some yeasts are effective biocontrol agent of post harvest diseases (Fan and Tian 2001; Janisiewicz and Korsten 2002) The use of was effective to control anthracnose of chili caused in storage (Chanchaichaovivat 2007). In addition, was also effective biocontrol agents against anthracnose, a post harvest disease of mango (Kefialew and Ayalew 2008). i.e. et al. Pichia guilliermondii by Colletotrichum gloeosporioides et al. Candida membranifaciens *Corresponding author, Phone/Fax: +62-251-8423048, E-mail: suryow@hotmail.com ISSN 1978-3477, eISSN 2087-8575 Vol 5, No 4, December 2011, p 154-159 I N D O N E S I A Available online at: http://www.permi.or.id/journal/index.php/mionline DOI: 10.5454/mi.5.4.2 B. theobromae B. theobromae cv. B. theobromae B. theobromae et al. Information on the use of yeast to control fruit rot of mango is not available. The objectives of this research were to investigate the potency of yeasts as biocontrol agent of fruit rot of mango caused by and to examine mechanism involved in the biocontrol. Twenty one yeasts isolates were used in this study. Four yeasts isolates (WSW1, WSW2, OSW1, and YSW1) were obtained from Plant Clinic of Institut Pertanian Bogor. Seventeen other yeast isolates were obtained from healthy mango fruit originated from Cirebon, West Java. Yeast isolation from fresh fruit of mango was done by skin washing than serial dilution technique up to 10 000 and continued by plating onto potato dextrose agar (PDA) pH 5.5. The isolated yeasts then purified and identified physiologically up to species level by using BIOLOG TM (Micro Log TM System, release 4.2). The pathogen was isolated from infected mango fruit by tissue plating technique and cultivated on PDA pH 5.5 and identified morphologically by based on Watanabe (2002). . Selection of yeast isolates for biocontrol effectiveness was conducted according to modified technique of Kefialew and Ayalew (2008). All of yeast were cultured on potato dextrose broth (PDB) pH 5.5 and shaken at 100 rpm for 3 days. Mango “Gedong Gincu” were obtained from farmer's field in Cirebon, West Java and kept under cool box and stored in refrigerator before handled. Mango fruits were disinfected by sodium hypochloride 1% for 1 min and rinsing by sterilized water and air dried. Thereafter mango fruit was dipped by yeast suspension at density of 10 cells mL for 1 min, then air dried. Skin fruit was taken up by knife up to size of 8 cm x 3 cm, put and placed into moistened 30 cm x 20 cm x 4 cm plastic pans. Preliminary research shows that disease severity produced by artificial inoculation of on mango fruit skin highly correlated to whole fruit inoculation. Then conidia of 7-day old of grown on PDA at the rate of 10 mL as 50 µL was pippeted on the yeast-treated mango skin, then mangos skin was incubated under dark for 24 h then incubated for 5 days. Disease severity was assessed by necrotic percentage of skin (Kefialew and Ayalew 2008). Biocontrol efficacy (BE) of yeasts was calculated using formulae of Chanchaichaovivat (2007) as follow: BE = (dc-dt)/dc x 100%; where BE, biocontrol efficacy (%); dc, disease severity of control; dt, disease severity of treatment. MATERIALS AND METHODS Yeast Used in This Study. Biocontrol Assay by o Testin-viv 6 -1 6 -1 Fruits dipping with sterilized water and fungicide with active ingredients thiram (Tiflo 80 WP) at the rate of 0.1% (w/v) for 1min were applied for negative and positive control respectively. The experiment was carried out using randomized complete designed with 4 replications. . Dual culture assay was performed to determine if biocontrol mechanism is antibiosis (Wisniewski 2007). All of yeasts isolates were tested against fruit rot fungi Yeast was streaked forming line in the middle of 9-mm petridish containing PDA. Agar plug Ø 3 mm-7- day old was seeded on the right and left of yeast streak, therefore line connecting two fungal colony centers was perpendicular to the yeast streak. Inhibition zone was assessed after 3 days of incubation and expressed in cm. The size of inhibition zone indicated antibiosis of yeasts against tested fungus (Spadaro 2002; Indriatmi 2008). All of treatments was arranged in randomized complete design and three times replicated. . All yeast isolates were grown on chitin agar, containing 0.5% colloidal chitin for 3 days (Shanmugaiah 2008). Chitinolytic activity was indicated by formation of clear zone surrounding yeast colony. Identification using BIOLOG resulted that there was variability of yeast originated from fructoplane of mango and Plant Clinic of IPB collection (from shallot's leaves). Four yeast isolates from plant clinic collection were identified as three species var. WSW1, var. WSW2, OSW 1. Only one isolate of plant clinic was a different species as from mango fructoplane YSW1. Moreover, seventeen yeast isolates from mango consisted of 11 species , var. and . Bioassay on biocontrol effectiveness showed that some yeasts isolates had ability to suppress fruit rot caused by (Table 2). Three isolates had disease biocontrol effectiveness rate over 35% which were var WSW1, K1 K 12 with biocontrol effectiveness as 70.83, 45.83 and 37.5%, respectively. Such effectiveness rate was comparable, even higher than standard fungicide Tiflo 80 WP with active ingredient thiram that had 35% (Table 2, Fig 1). Other important et al. B. theobromae. B. theobromae et al. i.e. Cryptococcus albidus aerius C. albidus aerius Candida edax i.e. C. terreus i.e. Candida edax, Candida mexicana, Candida terestre, Bulleromyces albus C. albidus aerius, Cryptococcus amylololentus, Cryptococcus l u t e o l u s , D e b a r y o m y c e s h a n s e n i i , P i c h i a guilliermondii, Rhodotorula aurentica, R. glutinis B. theobromae C. albidus . aerius P. guilliermondii , D. hansenii Dual Culture Assay Chitinolytic Activity RESULTS Volume 5, 2011 Microbiol Indones 155 finding was even though some isolates belong to the same species they had very different antagonistic activities. For instance, species var. only isolates WSW1 had high antagonistic activity (70.83%), other isolates WSW2, K6, and K 10 which had no antagonistic activity. The yeasts had variability in antibiosis indicating by the size of inhibition zone (Table 3). Some yeasts had strong antibiosis against such as var. . -dual culture test showed that var. WSW1, C. albidus aerius i.e. in-vitro B. theobromae C. albidus aerius In-vitro C. albidus aerius C. albidus WSW2, OSW1, YSW1, K2, K13, K6 had high antibiosis activity. Furthermore, other yeast isolates had lower antibiosis activity. Interestingly, there was no correlation between antibiosis activity and biocontrol efficacy. Among yeasts having high efficacy rate against fruit rot disease, only var WSW1 was effective yeasts and had high antibiosis activity. Other effective yeasts K1 and K7 had low antibiosis activity (Table 3). Ca. edax C. terreus C. luteolus Ca. edax C. albidus in vitro C. albidus . aerius P. guilliermondii D. hansenii Table 1 Identification of yeasts isolates using BIOLOG TM Table 2 Biocontrol effectiveness of yeasts against fruit rots of mango Isolates Origins Identified species Probability (%) WSW1 Plant Clinic (Shallot, Brebes) Cryptococcus albidus var. aerius 100 WSW2 Plant Clinic (Shallot, Brebes) Cryptococcus albidus var. aerius 100 OSW1 Plant Clinic (Shallot, Brebes) Candida edax 100 YSW1 Plant Clinic (Shallot, Brebes) Cryptococcus terreus 84 K1 Mango, Cirebon Pichia guilliermondii 94 K2 Mango, Cirebon Cryptococcus luteolus 91 K3 Mango, Cirebon Debaryomyces hansenii 99 K4 Mango, Cirebon Candida terestre 54.7 K5 Mango, Cirebon Candida edax 94 K6 Mango, Cirebon Cryptococcus albidus var. aerius 99 K7 Mango, Cirebon Bulleromyces albus 99 K8 Mango, Cirebon Rhodotorula glutinis 100 K9 Mango, Cirebon Pichia guilliermondii 99 K10 Mango, Cirebon Cryptococcus albidus var. aerius 99 K11 Mango, Cirebon Rhodotorula aurantica 99 K12 Mango, Cirebon Debaryomyces hansenii 98 K13 Mango, Cirebon Candida edax 99 K14 Mango, Cirebon Pichia guilliermondii 99 K15 Mango, Cirebon Candida mexicana 99 K16 Mango, Cirebon Cryptococcus amylololentus 84 K17 Mango, Cirebon Debaromyces hansenii 99 Isolate Codes Yeast species Disease severity (%) Biocontrol efficacy (%) WSW1 Cryptococcus albidus var. aerius 29.17 ± 5.13 g 70.83 WSW2 Cryptococcus albidus var. aerius 87.50 ± 4.54 b 12.50 OSW1 Candida edax 91.67 ± 4.37b 8.33 YSW1 Cryptococcus terreus 75.00 ± 5.81 cd 25.00 K1 Pichia guilliermondii 54.17± 4.69 f 45.83 K2 Cryptococcus luteolus 91.67 ± 6.53 b 8.33 K3 Debaryomyces hansenii 91.67± 6.53 b 8.33 K4 Candida terestre 95.83 ± 4.46 ab 4.17 K5 Candida edax 100.00 ± 0.00 a 0.00 K6 Cryptococcus albidus var. aerius 95.83 ± 4.46 ab 4.17 K7 Bulleromyces albus 95.83± 4.46 ab 4.17 K8 Rhodotorula glutinis 70.83 ± 6.72 d 29.17 K9 Pichia guilliermondii 75.00 ± 4.85 cd 25.00 K10 Cryptococcus albidus var. aerius 79.17± 4.48 c 20.83 K11 Rhodotorula aurentica 75.00 ± 4.35 cd 25.00 K12 Debaryomyces hansenii 62.51 ± 6.53 e 37.50 K13 Candida edax 70.83 ± 5.72 d 29.17 K14 Pichia guillermondii 91.67 ± 6.53 b 6.33 K15 Candida mexicana 87.50 ± 7.44 b 12.5 K16 Cryptococcus amylololentus 70.83± 6.53 d 29.17 K17 Debaryomyces hansenii 100.00 ± 0.00 a 0.00 Untreated (water) 100.00± 0.00 a - Fungicide a.i. Thiram 65.00 ± 7.11 e 35.00 Note: numbers followed by same symbols are not significantly different according DMRT test at α=0.05 156 SUGIPRIHATINI ET AL. Microbiol Indones Bulleromyces albus P. guilliermondii Among yeasts tested, only K7 and K 14 had chitinolityic activity (Table 3). Those two yeasts had low biocontrol efectiveness. Fructoplane and phylloplane are rich source of microbes, in which yeast is dominant group followed DISCUSSION Fig 1 Effectiveness of antagonistic yeasts against fruit rot disease of mango caused by in bioassay using mango skin. Note: A. K1, B. var. WSW1, C. Fungicide (thiram), D. Untreated Botryodiplodia theobromae Pichia guilliermondii Cryptococcus albidus aerius Code Species Inhibition zone (cm) Chitinolytic activity WSW1 Cryptococcus albidus var. aerius 2.10 ± 0.23a - WSW2 Cryptococcus albidus var. aerius 1.97± 0.16ab - OSW1 Candida edax 1.80± 0.12bc - YSW1 Cryptococcus terreus 1.83 ± 0.12bc - K1 Pichia guilliermondii 0.37 ± 0.11ij - K2 Cryptococcus luteolus 1.67 ± 0.12 c - K3 Debaryomyces hansenii 0.50 ± 0.21i - K4 Candida terestre 1.20 ± 0.26 de - K5 Candida edax 0.70 ± 0.33 j - K6 Cryptococcus albidus var. aerius 1.47 ± 0.18 d - K7 Bulleromyces albus 0.50± 0.18 i + K8 Rhodotorula glutinis 0.47 ± 0.19i - K9 Pichia guilliermondi 1.00± 0.10 fg - K10 Cryptococcus albidus var. aerius 1.17± 0.15 ef - K11 Rhodotorula aurentica 0.50±0.20 i - K12 Debaryomyces hansenii 0.40± 0.22 i - K13 Candida edax 1.43 ± 0.21d - K14 Pichia guilliermondii 1.03± 0.89 fg + K15 Candida mexicana 1.07 ± 0.25f - K16 Cryptococcus amylololentus 0.97±0.14 fg - K17 Debaryomyces hansenii 0.57 ± 0.14 i - Table 3 Antibiosis of yeasts against and their chitinolytic activityBotryodiplodia theobromae Note: + forming clear zone, numbers followed by same symbols are not significantly different according DMRT test at α=0.05 by bacteria and filamentous fungi (Elmer and Reglinski 2002). The number of isolated yeasts from fructoplane of mango in this study was 11 species , var. and . Some of isolated yeasts were potential as biocontrol agent i.e. var. i.e. Ca. edax, Ca. mexicana, Ca. terestre, B. albus C. albidus aerius, C. amylololentus, C. luteolus, D. hansenii, P. guilliermondii, R. aurentica, R. glutinis C. albidus aerius WSW1, P. Volume 5, 2011 Microbiol Indones 157 guilliermondii D. hansenii Pichia, P. anomala P. membranifaciens, P. guilliermondii et al Debaryomyces et al. C. albidus . aerius Cryptococcus Cryptococcus C. infirmo-miniatus C. laurentii C. laurentii et al. C. albidus C. albidus aerius Candida membranifaciens Ca. membranifaciens et al. C. albidus . aerius C. albidus . aerius, K1, and K12 indicated by having biocontrol efficacy rate over 35%. Yeasts belong to the genus such as , and have been reported as effective biocontrol agents against various post harvest diseases (Elmer and Reglinski 2001; Fan and Shiping 2002; Chancaichivat . 2008). Moreover sp. is also reported by lesser extent as biocontrol agent of post harvest diseases (McLaughlin 1990). The most effective yeast antagonist obtained in the study is var WSW1. Even though there are not many report on biocontrol using , some species of were reported as biocontrol agent, e.g., and against pear rot disease (Benbow and Sugar 1999), against blue mold of peach (Zhang 2007), and against blue and grey mold of apple (Fan and Tian 2010). It is interesting that one yeast isolate, var. WSW1 was very effective against fruit rot of mango (70.83% of biocontrol efectiveness) (Fig 1, Table 2), it was even higher than fungicide Tiflo 80 WP with active ingredients thiram that provide 35%. As comparison, research of Kefialew and Ayalew (2008) showed that the use as biocontrol yeasts on mango anthrachnose provided efficacy rate of 82%, on grey mold 61 % - 81 % (Gholamnejad 2010). This effectiveness of var WSW1 used in the study was very high and promising, because it was achieved under extreme disease conducive environment, in which diseases pressure is very high (conidia density 10 mL ), and incubated in moistened (100 % relative humidity) and darkened environment. In standard storage condition, the effectiveness is expected to be higher. Further test on combination of the yeast and real storage condition is necessary Other technique to increase efficacy are combining with other species, and or application and formulation optimization (Janisiewicz 1996). Based on data obtained in this experiment, even isolates WSW1, WSW2, K6, and K10 referred to species var they were very different in their antagonistic activity, only WSW1 had effective biocontrol activity. This shows that biocontrol effectiveness of tested yeasts is isolate-base and not species-base. Even though yeast isolates belong to same species, may have different antagonistic activity, therefore biocontrol activity test is necessary for each isolates. Mechanism of biocontrol is necessary to be recognized, therefore a biocontrol agent can further 6 -1 developed and optimized. There are various mechanism involve in biocontrol using yeasts - competition, antibiosis, lysis and resistance induction (Janisiewicz and Korsten 2001; De Ingeniis 2004; Wisniewski 2007). Weak correlation of antibiosis activity and biocontrol efectiveness such as depicted in Table 2 and Table 3, show that antibiosis is not main mechanism underlying biocontrol of yeasts. In this research, antibiosis is valid only for biocontrol mechanism of var. . Even though other previous researcher reported that chitinolytic and other lytic enzyme activity involve in mechanism of biocontrol using yeast (Spadaro 2002; Masih and Paul 2002), this study showed no relation between chitinolytic activity and biocontrol effectiveness of yeasts. Nutrients competition and induced resistance which involve in biological control control using yeast (Janisiewicz and Korsten 2001; El-Tarabily 2004; Yao and Tian 2005) were not investigated in this study. Further study to examine the role of induced resistance and competition of the three antagonistic yeasts is required. The study yielded three potential antagonistic yeasts effective against fruit rot of mango. Some of mechanism of antagonism has also been investigated. Thus one of important initial step in developing biocontrol agent has been carried out. Further study is needed to develop them as biocontrol agents: other mechanism involved, efficacy with real storage condition, environment and nutritional affecting factors, mass production and formulation technology. The first author would like to Indonesian Plant Quarantine Agency for providing opportunity and post graduate scholarship in Institut Pertanian Bogor, for financial and laboratory support for conducting the research et al. in-vitro C. albidus aerius . ACKNOWLEDGEMENT REFERENCES Brzezinska MS, Donderski W. 2001. Occurrence and activity of the chitinolytic bacteria of genus. Pol J Environ Stud. 10(1):27-31. Benbow JM, Sugar D. 1999. Fruit surface colonization and biological control of postharvest diseases of pear by preharvest yeast applications. Plant Dis. 83 (9):839-844. Chanchaichaovivat A, Ruenwongsa P, Panijpan B. 2007. Screening and identification of yeast strains from fruits and vegetables: potential for biological control of postharvest chili anthracnose ( ). 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