02. Putranto.cdr Vol.15, No.3, September 2021, p 84-90 DOI: 10.5454/mi.15.3.2 Are and Effective to Control Trichoderma atroviride Trichoderma harzianum Fusarium Associated ith Tomato Wilt?w WILFRIDUS ADYATMA PUTRANTO , RULLY ADI NUGROHO , PETRUS SUNU 1 1* HARDIYANTA DESTI CHRISTIAN CAHYANINGRUM 2 1 , AND 1 Faculty of Biology, Satya Wacana Christian University, Jalan Diponegoro 52-60 Salatiga 50711 Indonesia; 2Faculty of Pharmacy, Sanata Dharma University, Jalan Affandi, Mrican Yogyakarta 55281 Indonesia. The pathogenic fungi, such as in the rhizosphere of tomato ( ) negatively Fusarium Solanum lycopersicum affects the yield and quality of the plant. A number of biological control agents have been used for protecting tomato plants against wilt diseases including various fungal species. The objective of this study was to evaluate the antagonism effects of sp. associated T atroviride T. harzianum Fusariumrichoderma and against the pathogen with tomato wilt In this study, the antagonism of these spp. against the sp. was tested . Trichoderma Fusarium in vitro by the dual culture technique, and the percentage inhibition of radial growth (PIRG) and the antagonism reaction (scale 1-5) were evaluated. The results showed that . and . led to 70.8% PIRG and T atroviride T harzianum scale 1 antagonism reaction, and 40.6% PIRG and scale 3 antagonism reaction against sp. associated Fusarium with tomato wilt after 7 days of incubation, respectively. These results indicate that application of . and T atroviride T harzianum Fusarium. may be promising approach for biological control of wilt of tomato and may play an important role in sustainable agriculture. biological , , tomato, Key words: antimicrobial activity, control wilt spp. F Tusarium richoderma Jamur patogen, seperti di rizosfer tomat ( ) berdampak negatif terhadap hasil Fusarium Solanum lycopersicum dan kualitas tanaman. Sejumlah agen pengendali hayati, termasuk berbagai jenis jamur telah digunakan untuk melindungi tanaman tomat dari penyakit layu. Penelitian ini bertujuan untuk mengevaluasi dampak antagonisme Trichoderma atroviride T harzianum Fusarium and . terhadap patogen sp. penyebab layu pada tomat. Dalam penelitian ini antagonisme spp. terhadap sp. diuji secara in vitro dengan teknik kultur Trichoderma Fusarium ganda, dan persentase penghambatan pertumbuhan radial (PPPR) dan reaksi antagonisme (skala 1-5) dievaluasi. Hasil penelitian menunjukkan bahwa . dan . masing-masing menyebabkan 70,8% PPPR T atroviride T harzianum dan reaksi antagonisme skala 1, dan 40,6% PPPR dan reaksi antagonisme skala 3 terhadap sp. penyebab Fusarium layu pada tomat setelah 7 hari inkubasi. Hasil ini mengindikasikan bahwa aplikasi . dan . T atroviride T harzianum dapat menjadi pendekatan yang menjanjikan untuk pengendalian hayati layu tomat dan dapat Fusarium memainkan peran penting dalam pertanian berkelanjutan. Kata kunci: aktivitas antimikroba, pengendalian biologi, layu , tomat, spp.Fusarium Trichoderma MICROBIOLOGY INDONESIA Available online at http://jurnal.permi.or.id/index.php/mionline ISSN 1978-3477, eISSN 2087-8575 *Corresponding author: Phone 62-298-321212 ; : + Fax: +62- 298-321433; rully.nugroho@uksw.edu E-mail: Fusarium become harmful organisms for tomato plants (Solanum lycopersicum) because it will cause tomato plants to be damaged and harvest failure. Tomato plants that are attacked by will turn yellow on the F. oxysporum leaves, the plants will wither, growth will be stunted, the fruit will rot, then the plant will die and necrosis on one side of the stem. Symptoms of necrosis begin with a change in color, then gradually dry up (Wiam et al. 2019). Seedlings exposed to f. sp. F. oxysporum lycopersici experience slow growth, while plants infected with cause root rot partially or Fusarium completely, leaves turn yellow and then curl, plants are stunted, wilted, and may die completely (Blancard 2012; Hermann and Lecomte 2019). can Fusarium produce mycotoxins tricotisin, fumonisin, intentin, zearalenone, beauverisin, moniliformin, fusarin, fusaric acid, and fusaproliferin affect human and which can animal health (Wang 2011).when ingested et al. Fusarium is one of the most economically important genera of fungal plant pathogens, causing significant crop losses and contamination of grain by mycotoxins on a global basis (Burgess and Bryden 2012). The species most commonly involved include F. graminearum F. oxysporum et al. and (Dean 2012). Fusarium colonizes the xylem vessels producing mycelium and conidia. The characteristic wilt symptoms appear as a result of water stress, mainly due to vessel clogging (Beckman 1987). Fusarium wilt of tomato is commonly caused by the fungal pathogens, . , . or . F oxysporum F solani F equiseti (Isaac . 2018; Ayele . 2021). The most familiar et al et al formae species to tomato is f.sp. Fusarium oxysporum lycopersici et al et al (Isaac . 2018; Srinivas . 2019). Farmers use chemical fungicides such as benomyl, mancozeb, , and dithiocarbamate phenylamide to treat problems caused by pathogenic fungi (Apriani . et al 2014; Sumardiyono . 1995; et al Worku and Sahela 2018). However, long-term use of fungicides can affect non-target organisms such as earthworms, microbes and humans (Patel 2014). Biological control et al. technology plays an important role in overcoming diseases in plants that are promising and safe for humans and do not pollute the environment (Kang 2019). Currently there are more than 60% of , biological control species that use (Abbey Trichoderma et al. 2019). can be found at Trichoderma species temperatures of 25-35°C (Hajieghrari 2008) in et al. tropical soils, and can also be found on soils in forest, agricultural, and grassland areas (Kubicek 2003). et al. As many as 35 of the 260 live Trichoderma species that in roots (Digamber 2017) have good economic importance because of their ability to produce enzymes, antibiotics, and secondary metabolites so that they can be used as effective biological control agents (Blaszczyk 2014). is known as a et al. Trichoderma biological control agent because it can survive in all kinds of conditions, has a high reproductive rate, is efficient in the absorption of nutrients from its surroundings, and shows aggressive resistance to a fungal pathogen (Misra and Prasad 2003 Pandya ; et al. 2011). Zhang (2017) stated that showed et al. Trichoderma antagonistic behaviour against several phytopathogenic organisms, including bacteria, nematodes and fungi by inhibiting their growth. (Jiang T asperellumrichoderma et al. T. harzianum et al. T. 2016), (Ezziyyani 2007), koningiopsis et al. T. virens (Delgado 2018), and (Tomah 2020) have been shown to be highly et al. effective in the management of . Phytophthora capsici According to Jiang (2016) hyphae from isolates of et al. T. asperellum P. can penetrate hyphae and spores of capsici in the form of mycoparasitism which causes hyphal cells to degrade. Mycoparasitism involves cell wall-degrading enzymes that allow mycoparasitic fungi to drill holes into other fungi and extract nutrients for their own growth (Cao 2009). strains et al. Trichoderma produce antibiotics or low molecular weight compounds that are useful for inhibiting the growth of plant pathogens such as 6-pentyl-pyrone (Jelen et al. 2014), viridiofungin (El-Hasan 2009), and et al. gliotoxin (Roberts and Lumsden 1990). as an interesting model for being Trichoderma studied about interactions between plant hosts and its symbionts should be as aconsidered biological control alternative in the green economy era which aims to maintain human health, protect the environment, and as a promotional agent sustainable agriculture (Lopez- Bucio 2015; Guzmán 2017; Sood et al. et al. et -Gusmán al. 2020). Therefore, this study was conducted to test whether can overcome the T. atroviride T. harzianumor pathogen tomato so that Fusarium associated with wilt these two species can be used as Trichoderma biocontrol agents. MATERIALS AND METHODS Isolation of ungal trains. F S The Trichoderma atroviride T. harzianum and isolates were obtained from , Salatiga, KPTT Agricultural Training Center Indonesia and recultured in Potato Dextrose Agar (PDA Merck). isolates were , spp. Trichoderma incubated at 25°C for 7 days (Ramteke 2019). The patogen, sp. was isolated according to a Fusarium procedure developed by and (Leslie Summerell 2006) from the wilt infected roots of tomato plants which were collected from KPTT Agricultural Training Center . grown , Salatiga, Indonesia The isolates were on Komada's selective medium and incubated at 30°C for 7 days, then PDA and subcultured on (Merck) incubated at 25°C for 5 days Leslie Summerell ( and 2006 . ) sp. isolate was identified on the basis Fusarium of cultural and morphological characteristics which included colony color, pigmentation, surface texture, colony edge, conidiophores, septate or anseptate hyphae, presence or absence of phialid, conidia color, conidia shape, macrocoonidia or microconidia shape, presence or absence of chlamydospores (Mwaniki et al. et al. 2011; Sharma Singh 2014; Rai 2016; and Redda 2018). et al. The In vitro xperiment. E effects of Trichoderma spp. on were tested by the dual culture Fusarium technique as described by The Redda (2018). et al. Trichoderma Fusarium spp. isolates and sp. to be tested were cultured separately on PDA for 7 days. After 7 days, 5 mm mycelial plugs (taken from the edge of fungal colonies) of each species to be tested were transferred to PDA plates using cork borer. The mycelial plug of species and Trichoderma Fusarium sp. was placed opposite side to each other on a PDA surface. PDA plates inoculated with sp. was Fusarium included as negative control. All plates were incubated at 25 2 C and observations were made after 7 days of + ° incubations. The percentage inhibition of radial growth (PIRG) was calculated by the following formula Volume 15, 2021 Microbiol Indones 85 86 PUTRANTO ET AL . Microbiol Indones ( ):Sharfuddin and Mohanka 2012 PIRG = (R - R )/R1 2 1×100 where radial growth of sp. in control R = 1 Fusarium (cm), radial growth of sp. in dual culture R = 2 Fusarium tests with spp. (cm). The antagonism Trichoderma reaction of isolates were score using a Trichoderma scale of 1 to 5 after 7 days of incubation, where 1 = Trichoderma gr over ew the entire growth of the Fusarium sp., over ew at -2 = gr least twoTrichoderma third of the medium 3 = and surface, Trichoderma Fusarium sp. each one colonized half of the medium surface, sp. colonized at least two-third 4 = Fusarium of the medium surface, sp. overgrew5 = Fusarium Trichoderma ( ).Bell . 1982et al Statistical .Analysis were analysed using Data SPSS 22 (SPSS Inc., Chicago, IL). All data were calculated and analysed statistically using test (t P < 0.05) to determine differences between treatments. The table was drawn using Microsoft Excel 2016. RESULT Isolation and dentification of sp. I Fusarium Results showed that sp. had well-defined Fusarium macroscopic characteristics (Fig 1). Fusarium sp. has pink mycelia in the center and white at the edges. Fusarium sp. underwent pigmentation changes where the mycelia of sp. on the first and second day Fusarium it is white and on the fifth day it changes color to a slightly brownish beige. In addition, the surface texture of sp. has a cotton-like texture and has a Fusarium filamentous shape and colony edge. Conidiophores Fusarium sp. are dendritic conidiophores, possessing pseudohyphae, but no phialids. Color of conidia Fusarium sp. is a fusoidal green with thickened walls at the ends. ha chlamydiophores that are Fusarium s shaped like lumps on hyphae. The Effects of T Fusariumrichoderma spp. on sp. using AssaysIn Vitro . Table 1 showed the PIRG observed on the first until the seventh day of the test. The inhibitions of . and . are T atroviride T harzianum observed from the second and the third day of incubation, respectively. Both isolates were shown to have inhibitory activity on pathogen in vitro Fusarium (Table 1). was significantly ( Trichoderma atroviride P < 0.05) more effective as it was able to inhibit at 70.8% of sp. after 7 days of incubation, while . Fusarium T harzianum demonstrated lower PIRG (40.6%) after 7 days of incubation. In Fig 2, a 7 day test of sp. Fusarium grown with . and . clearly T atroviride T harzianum demonstrated that the mean scale for antagonism of . T atroviride T was also significantly higher than . harzianum, i.e. scale of 1 and 3, respectively. DISCUSSION The isolate obtained from tomato rhizosphere soil was confirmed as sp. The morphological Fusarium characteristics are the same as mentioned by Gordon (2017) and Srinivas . (2019). The result of this et al study supports findings in other studies that characterized as most important fungal in Fusarium tomato plantation areas in defferent countries of the world (Grattidge and O'Brien 1982; Jones . 1991; et al Steinkellner . 2005; Rozlianah and Sariah 2006; et al Amini 2009; Chehri 2016). fung that the Trichoderma species are i have potential to control pathogenic fungi, has a low impact on soil balance, and has minimal impact on damage to non-target organisms (Sood 2020).et al. Consistent with previous findings, this study confirms the antagonism effect of .T atroviride T. harzianum and against the pathogen Fusarium sp. associated with tomato wilt. The antagonism effects of both Trichoderma spp. sp. showed that against Fusarium both spp. affecting the development Trichoderma pattern of sp. colonies. Fusarium Trichoderma colonies presented a faster growth in the plates of dual cultures, being capable of growing on the Fusarium sp., preventing their mycelial development by nutrient and space competition as found by Filizola . et al (2019). Moreover, based on the antagonistic activity (Soytong 1988), this study indicated that . T atroviride exhibited a very high antagonistic activity against Fusarium T harzianum sp. (>75% PIRG), while . Table 1 The percentage inhibition of radial growth (PIRG) in dual culture assay. Results are expressed as mean standard deviation of PIRG after 1- to 7-day incubation. ND = Not Determined+ Trichoderma strains 1-day 2-day 3-day 4-day 5-day 6-day 7-day T. atroviride ND 2.9 ± 11.94 36.5 ± 6.50 51.7 ± 3.39 60.5 ± 2.44 66.7 ± 2.34 70.8 ± 2.03 T. harzianum ND ND 5.0 ± 8.69 6.1 ± 7.08 21.3 ± 5.67 32.3 ± 4.46 40.6 ± 3.76 Percentage inhibition of radial growth (PIRG) (%) Volume 15, 2021 Microbiol Indones 87 Fig 1 Characterization of sp. by macroscopic morphology in PDA medium after 7 days. a. Colony Fusarium morphology; b. Conidiosphores and conidia. Magnification 400× . Fig 2 Dual culture assay after 7 days of incubation. a. / sp.; b. Trichoderma virid Fusarium Trichoderma atro e harzianum Fusarium Tv Trichoderma atroviride Th Trichoderma harzianum F Fusarium/ sp.; : ; : ; : sp. showed low antagonistic activity against sp. Fusarium (<50% PIRG). This study shows that . T atroviride demonstrated more antagonistic activity than observed by Stracquadanio . (2020). Stracquadanio . et al et al (2020) found that . did not reduce the T harzianum growth of . This shows that not Fusarium moniliforme all strains are able to inhibit the in vitro Trichoderma growth of .Fusarium Previous studies have shown that has Trichoderma several modes of action, including production of antibiotics and cell wall degrading enzymes, competition for key nutrients, parasitism, stimulation of plant defense mechanisms and combination of these possibilities (Fravel 1988; Larkin and Fravel 1988; Roberts and Lumsden 1990 El-Hasan 2009; ; John et al. et al. 2010; ; ; Taghdi Jelen 2014 Jiang 016 et al. et al. 2 et al et . 2015). According to Howel (2006) and Zhang al Trichoderma. (2017), exhibit antagonistic behavior against several phytopathological organisms, especially fungi by inhibiting their growth either by direct interactions (e.g. hyperparasitism, competition for nutrient and space, and antibiosis). 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