BIODIVERSITAS ISSN: 1412-033X Volume 24, Number 7, July 2023 E-ISSN: 2085-4722 Pages: 3844-3852 DOI: 10.13057/biodiv/d240723 Isolation of endophytic fungi from Hiyung chili peppers of local South Kalimantan (Indonesia) varieties and in vitro tolerance to acidic environment WITIYASTI IMANINGSIH 1,2, , NURAENI EKOWATI 2 , SALAMIAH 3 , NUNIEK INA RATNANINGTYAS 2 , LOEKAS SOESANTO 4 1 Microbiology Laboratory, Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Lambung Mangkurat. Jl. A. Yani Km 36.4, Banjarbaru 70714, South Kalimantan, Indonesia. Tel./fax.: +62-511-4773112,  email: witiyastiimaningsih@ulm.ac.id 2 Faculty of Biology, Universitas Jenderal Soedirman. Jl. dr. Suparno No. 63, Purwokerto Utara, Banyumas 53122, Central Java, Indonesia 3 Department of Plant Protection, Faculty of Agriculture, Universitas Lambung Mangkurat. Jl. A. Yani Km 36.4, Banjarbaru 70714, South Kalimantan, Indonesia 4 Faculty of Agriculture, Universitas Jenderal Soedirman. Jl. dr. Suparno No. 61 Grendeng, Purwokerto Utara, Banyumas 53122, Central Java, Indonesia Manuscript received: 19 April 2023. Revision accepted: 14 July 2023. Abstract. Imaningsih W, Ekowati N, Salamiah, Ratnaningtyas NI, Soesanto L. 2023. Isolation of endophytic fungi from Hiyung chili peppers of local South Kalimantan (Indonesia) varieties and in vitro tolerance to acidic environment. Biodiversitas 24: 3844-3852. Hiyung chili (Capsicum frutescens L) is a local chili from Hyung Village, Tapin District, South Kalimantan Province, Indonesia which has the advantage of being cultivated with an agricultural system in swampland, with high production and is known to have high capsaicin also. This advantage is thought to be related to its symbiosis with endophytic microbes, especially fungi. The purpose of this study was to isolate and identify non-pathogenic endophytic fungi from Hiyung chili plants and determine their ability to tolerate acidic conditions in vitro. Results showed that a total of 41 endophytic isolates were isolated from different parts of chili plant. Of the 41 isolates four non-pathogenic endophytic fungi were isolated from leaf tissue. Based on morphological and molecular identification these four fungi were Pseudozyma hubeiensis DM3.1, Phoma sp. DT2.2P1, Letendraea sp. DM1.2 and Phoma sp. DT2.2P4. Four endophytic fungi showed different abilities in an in vitro preliminary study of acidic environmental stress tolerance. Only P. hubeiensis DM3.1 isolate had the daily diameter of colony not significantly different based on the Anova test (α :0.05, F: 1.092, P:0.372) both at pH 5, 4 and 3 and without pH treatment. Keywords: Acid stress, endophytic fungi, Hiyung chili local variety INTRODUCTION Hiyung cayenne pepper is one of the peppers (Capsicum frutescens L.) grown by farmers in South Kalimantan (Wahdah et al. 2016; Hamdani et al. 2017). These chilies are cultivated in the swampland of Hiyung Village, Tapin District and have been registered as a protected variety and also designated as a national variety by the Ministry of Agriculture of the Republic of Indonesia (Pramudyani et al. 2019) and have received a certificate of geographical indication by Ministry of Law and Human Right Republic of Indonesia at 2019 (Rochman and Tarmizi 2020). The superiority of cayenne pepper is listed in the attachment to the Decree of the Minister of Agriculture no. 031/Kpts/SR.120/D.2.7/4/2016 namely high production (6 to 7 tons per hectare) with a high number of fruits per plant (1820-2180), with a very spicy taste (capsaicin: 2333.05 ppm) and long shelf life (10-16 days at room temperature) (Hamdani et al. 2017). The tolerance of these chilies to grow in swamplands is high, its production is also better when compared to those grown in the dry land. The swampland where Hiyung chili is cultivated is acidic, with a pH of 5 (Pramudyani et al. 2019). The ability of plants to adapt to the environment may be related to their relationship with endophytic microbes (Giauque et al. 2019). Some microbes, both bacterial and fungal, are known to live in plant tissues as endophytes. Endophytes live internally, are associated in healthy plant tissues and their presence does not interfere with plant growth (Rajamanikyam et al. 2017) and does not cause disease symptoms (Mishra et al. 2014). Most endophytic fungi also live as saprophytes in the environment around plants (Wenndt et al. 2021). Fungi are more commonly found in environments with acidic conditions, thus endophytic fungi are easy to find on plants living in acidic swamps because they can adapt well as help plants to survive with these conditions (Vylkova 2017). Several endophytic fungi have been isolated from Hiyung chili. Trichoderma sp. ACH1.1, Trichoderma sp. ACH1.6, Trichoderma sp. ACH2.2, Botrytis sp. ACH2.3, Gliocladium sp. ACH2.4, Harmoniella sp. ACH2.5, Humicola sp. ACH2.6, Cunninghamella sp. ACH2.7 is entirely derived from the root (Imaningsih et al. 2021) and Trichoderma sp. DN3, Trichoderma sp. AK2), Trichoderma spp. BT1 derived from the root, leaves, and stems of Hiyung chili (Budi and Mariana 2016). These endophytic fungi are known to be able to inhibit the growth