Identification of Simplicillium lanosoniveum at suppressive soil potential areas in Brantas watershed – Indonesia Cite as: AIP Conference Proceedings 2583, 020032 (2023); https://doi.org/10.1063/5.0116197 Published Online: 13 January 2023 Ambar Susanti, Primaadi Airlangga and Ino Angga Putra AIP Conference Proceedings 2583, 020032 (2023); https://doi.org/10.1063/5.0116197 © 2023 Author(s). 2583, 020032 Identification of Simplicillium lanosoniveum At Suppressive Soil Potential Areas In Brantas Watershed – Indonesia Ambar Susanti1, a), Primaadi Airlangga2 , and Ino Angga Putra 3 Department of Agroecotechnology, KH.A. Wahab Hasbullah University, Jombang, Indonesia Department technology of informatics, KH.A. Wahab Hasbullah University, Jombang, Indonesia 3 Department of physical education, KH.A. Wahab Hasbullah University, Jombang, Indonesia 1 2 a) Corresponding author: sekarsasanti@gmail.com Abstract. Simplicillium lanosoniveum strain CG888 (MT 081944.1) is one of the fungi was isolated from the results of rhizosphere soil exploration in Gondang Manis Jamaica apple (Syzygium malaccense L), Perak District, Jombang Regency, Indonesia, which is included in the Brantas watershed area. Sampling was conducted in July 2020, using a one diagonal sampling method with five sample points. Purification of fungal isolates from soil samples was carried out using the dilution method, up to a level of 10-2. The DNA extraction results were amplified using the PCR technique. ITS4 and ITS5 primers were used for the amplification of the ITS rDNA region. The sequencing results were analyzed based on GenBank data at the National Center for Biotechnology International (NCBI). using the Basic Local Alignment Tools (BLAST) program. The phylogenic structure was determined using MEGA 7.0 software. Based on macroscopic, it has white colonies on the upper surface, and the lower is yellowish. Yellowish color is found in the old colonies or the center of the colony, and sphere. The structure is like cotton with a thick mycelia arrangement. Microscopically, The hyphae has transparent and septate, branching, tapering to the tip, and at the end formed phialid. The conidia are transparent, small and in round shape. Simplicillium lanosoniveum strain CG888 (MT 081944.1) as one of the antagonist fungi among other fungi that have been found and identified, so that it can support soil in the Brantas watershed area has the potential as a suppressive soil. INTRODUCTION The Brantas Watershed area has a fairly good irrigation system. The water that passes through the Brantas watershed comes from Mount Arjuno, then crosses Mount Kelud and flows into lowland areas which have an altitude of 25-100 m above sea level, with a slope of 0-20%. The damaged conservation areas in the upstream part, the increased of agricultural cultivation, and the erosion in the highlands in the rainy season accompanied by high rainfall, resulting in a lot of topsoils, accumulation of pesticides, and agricultural residues being carried away by river currents and deposited in lowland watersheds. This allows a high variety of types and quantities of land carried and deposited into the watershed. there are several types of soils generally in the watershed area, including andosol, grumosol, and lithosol1. The andosol soil generally has a hollow structure and is an ideal place for plant root growth, besides that it also has a fairly good content of organic matter2. Meanwhile, on the other hand, grumosol soil contains elements of low N, P, K, and organic matter3,4. Hardjowigeno [5] said that the same thing is also found in the characteristics of lithosol soil, which is a relatively young soil type, which is less than 10 cm from the soil surface, with a rough soil texture that tends to result in minimal nutrient elements, because it cannot bind these elements in the soil Simatupang [6] reported that the rhizosphere is an environment that has large quantities and diverse populations of microorganisms. The Association of microorganisms in the yard and plant roots has various roles. These roles include helping in the formation process of soil structure, nutrient cycling, and the activity of microorganisms that are detrimental or beneficial to a plant’s growth and development. Furthermore, The presence of microbes is more in the rhizosphere region and has differences in quality and quantity in different regions. bacterial and fungal The 5th International Conference on Agriculture and Life Science 2021 (ICALS 2021) AIP Conf. Proc. 2583, 020032-1–020032-7; https://doi.org/10.1063/5.0116197 Published by AIP Publishing. 978-0-7354-4216-0/$30.00 020032-1 populations were found ranging from 106 - 109 and 105 - 108, respectively, in one gram of soil in the rhizosphere7. These are the factors that make lands in the area become potential as suppressive soil. Based on the results of exploration that had been carried out in the Rhizosphere of Gondang Manis Jamaica apple (Syzygium malaccense L) plantation, Perak Subdistrict in July 2020 during the dry season with rainfall ranging from 1001 - 1500 mm, the explorer obtained Mycorrhiza sp., Trichoderma sp., Beauveria sp., Fusarium sp., Metarhizium sp., Aspergillus sp., Penicillium sp., and one fungus that has not been identified. It is necessary to carry out an identification test for the unidentified fungi, to determine whether it has the potential to be antagonistic or entomopathogenic fungi. Hopefully, this can support the soil in the Brantas watershed area as a suppressive soil with the diversity of antagonistic fungi in the area. THE MATERIALS AND METHOD Location and Sampling The unidentified fungi were taken from the exploration of rhizosphere soil in Gondang Manis Jamaica apple (Syzygium malaccense L) plantation, Perak sub-district, Jombang district. Sampling was conducted in July 2020, using a one diagonal sampling method with five sample points. The soil is taken weighing 1-2 kg, at a maximum depth of 30 cm from the ground surface. The soil was then composited and put into a plastic bag. Then, it was taken to the Laboratorium of the Agriculture’s Faculty KH.A. Wahab Hasbullah University. Fungi Isolate Purification Purification of fungal isolates from soil samples was carried out using the dilution method8. One gram of soil was dissolved with 10 ml of aquadest, using a magnetic stirrer with a speed of 500 rpm for 15 minutes. Then the dilution was carried out in a 10 ml test tube consisting of 1 ml dilution and 9 ml aquadest. The dilution was stratified, each level was homogenized with vortex, and 1 ml of each level of dilution was taken, which was then added with 9 ml of distilled water until the level of dilution was 10-2. After that, the solution was poured on the surface of the PDA media in a petri dish and flattened. Pouring was carried out under sterile conditions inside the LAF. Then it was incubated at room temperature 27-29oC for 7 days. Fungi Identification Observation After the fungi appear on PDA media, the target fungi are separated from other fungi. The fungi were purified by replanting them in sterile conditions on PDA media (PDA, 1% w / v peptone) in a petri dish and incubated for up to 14 days. Observations were made on the growth and development of fungal colonies, identification of macroscopic and microscopic morphology. Microscopic observations were made using an Olympus cx 31 microscope (Olympus Corporation, Tokyo Japan). Fungi were also cultured in potato dextrose broth (PDB) medium for molecular testing. Molecular Test The results of fungi culture from PDB were taken as much as 0.5 grams for DNA extraction, using 2% CTAB (sterile distilled water, Tris HCl 1M, EDTA 0.5 M, NaCl 5M, CTAB, B-mercapto-ethanol 1%,). Furthermore, the DNA extraction results were amplified using the PCR technique based on the procedure of Curran et al. [9]. ITS4 and ITS5 primers were used for the amplification of the ITS rDNA region. water 9,5 µl, and PCR kit 12,5 µl. 1% agarose is used for electrophoresis as a result of amplification. Then it is immersed in TBE 1x which has been mixed with 2 µl of Ethidium bromide. After that, the 5 µl PCR DNA sample is inserted into the well, 100bp and 1 kb were inserted into the well as much as 5 µl. DNA measurements were carried out using a DNA ladder of 100 bp and 1 kb. Electrophoresis was carried out with 50 volts for 50 minutes (in 0.9% agarose). The results were presented using an ultraviolet transilluminator (MaestroGen Inc., Hsinchu City Taiwan) and documented using Gel Documentation. Then the samples were sent to FirstBase Malaysia. The sequencing results were analyzed based on GenBank data at the National Center for Biotechnology International (NCBI). using Basic Local Alignment Tools (BLAST) program. After obtaining BLAST results, the phylogenic structure was determined using MEGA 7.0 software. 020032-2 RESULTS AND DISCUSSION The result of incubation of the rhizosphere soil dilution suspension from the Gondang Manis Jamaica apple (Syzygium malaccense L) planting, the target fungi were obtained. After separating from other fungi and incubating them after 25 days, a display of the growth and development of target fungi is obtained as shown in Figure 1 below. FIGURE 1. Macroscopic cross-section of purified target fungi 25 days after incubation (a) Upper colony surface, (b) Lower colony surface The color of the colonies on the upper surface was white, while on the lower surface it was yellowish. The yellowish color is found in the old part of the colony or the middle of the colony. The color changes that occur on the lower surface of the colony are initially white. As it grows it forms a sphere, with the center turning yellow. The edges are white with new hyphal growth. Figure 1 shows the colony texture like cotton with a thick mycelial arrangement. The upper surface is wavy like a pile of cotton, while the lower surface is wrinkled around the old colony. The morphological characteristics of the fungi above are following the previous report that the media of the Simplicillium lanosoniveum isolate from their research had white mycelia, while the bottom of the media was creamy yellow10. The results of research on the Simplicillium genus is also reported that the colonies of species in this genus generally grow fast, with a period of 10 days ranging from 10–38 mm, white on PDA media, while the bottom of the cream turns pale yellow, with a cotton-like edge, smooth11. FIGURE 2. Microscopic of target fungi hyphae from rhizosphere exploration in Gondang Manis Jamaica apple (Syzygium malaccense L) plantations; (a) structure of target fungi hyphae assemblages, (b) microscopic of hyphae characteristics; (1) phialids, (2) septa hyphae, (c) Conidia of target fungi Figure 2 shows the results of microscopic observations of these fungi, which are known as transparent and septic hyphae. Branched hyphae with orderly branching locations (Fig. 2a). The morphology of the branches of the hyphae is tapered, and at the tip are phialids formed (Fig. 2b). Phialids produce conidium attached to the tip. Based on the results of microscopic morphological observations, the target fungal hyphae according to the description of the Simplicillium fungi, namely thin and hyaline mycelium, septic and branched, phialides arise from the tip of the hyphae tapering towards the apex, elongated, slender, and walled smooth17. Figure 2(c) shows transparent conidia, small in size and round in shape. Generally, the conidia of Simplicillium fungi are formed into small, round heads, which are formed sometimes in zigzag or imbricate chains, branched, or unbranched 17 . 020032-3 Further identification using PCR analysis of these target fungi. The results of PCR analysis are shown in Figure 3 indicated by a sign of the presence of a band that identifies the isolate as successful, therefore it can continue to DNA sequencing process. FIGURE 3. Visualization of amplified DNA bands using ITS4 and ITS5 primers. M.Marker 1kb and 100 bp DNA Ladder from target fungi. Based on the universal primers ITS4 and ITS5 used in DNA extraction and the sequencing process that produce phylogenic (Fig. 4), they indicate that the identified fungi is Simplicillium lanosoniveum strain CG888 (MT 081944.1). FIGURE 4. Phylogenic pyramid from DNA extraction and sequencing Simplicillium lanosoniveum strain CG888 (MT 081944.1) is one of the fungi that have been explored in the rhizosphere of Gondang Manis Jamaica apple (Syzygium malaccense L) plantations. Characteristics and distribution of the fungi Simplicillium lanosoniveum to its host are in the form of saprophytes in fungi, endophytes, pathogens in plants, parasites in nematodes and insects, as well as their soil habitat11. This fungi is included in Cordycipitaceae, and the anamorphic of genera Beauveria and Isaria 12,13,14. Furthermore, Simplicillium and Lecanicillium are Verticillium spp formerly 12,13,14, and both of them are as Ascomycetes that expressed in mycoparasitic and entomophatogenic 14 . The reports on the interaction of S. lanosoniveum with other microorganisms are still limited, aspecially in Indonesia. It had a potential mycoparasitic to control Aecidium elaeagni-latifoliae that caused rust at Elaeagnus latifolia 15. Moreover, S. lanosoniveum could reduce the development of Phakopsora pachyrhizi that caused rust diseases at soybean 14. Based on S. lanosoniveum is entomopathogenic, the results of the research showed that onethird of Bombyx mori larvae failed to become pupae, which resulted in an incomplete metamorphosis cycle due to S. lanosoniveum infection16. It was able to cause the death of Coccus hesperidum larvae 20 days after inoculation16. Liu Cai, Zhao, et.al [15] stated that these fungi are also the plant parasite nematode pathogens. Simplicillium. lanosoniveum Cs0701 had high virulence against plum plant aphids Hysteroneura setariae with an average mortality 020032-4 rate of 86.33 % compared to controls (21.33%) five days after inoculation17. It was able to inhibit the growth of powdery spores mildew 14% in Murraya paniculata citrus plant compared without treatment (55.17%) 17. The existence of S. lanosoniveum strain CG888 in the rhizosphere of Gondang manis Jamaica apple (Syzygium malaccense L) is related to the factors that influence it. Carlile et al. [18] stated that soil in the rhizosphere generally has a population of microorganisms that is more and more diverse than in non-rhizosphere soils. The diversity and population of microorganisms are also carried and deposited in the rhizosphere layer, including S. lanosoniveum. In addition, Patra et.al. [19] reported that the movement of nutrients in the soil is influenced by several factors including physical, chemical, biological, environmental (temperature and humidity), and the types of plants that grow. Furthermore, Patra et.al. [19] said that these factors affect the population of microorganisms in the soil. Conklin [20] reported that in one gram of soil there are 108-109 types of bacteria, 107- 108 species of actinomycetes per gram of soil, 105-106 fungi propagules per gram of soil. These microbes (bacteria, actinomycetes, and fungi) are quite numerous in soils. The development of a diverse population of microorganisms in plant roots is influenced by the exudate released by plant roots 21. Badri and Vivanco [22] states that the number and types of root exudates released are determined by the type and age of the plant, as well as biotic and abiotic environmental factors. Initiated by unfavourable soil conditions for plant growth and development, plants are encouraged to survive in soil and environmental conditions, one of the methods used is by releasing root exudates into the soil. The roots release exudate as a form of defense against the stress of the surrounding environment. The role of the host tree in the abundance of fungal spores in the rhizosphere is caused by root exudate it produces, then the root exudate as a source of energy will affect the germination of fungal spores 23. Microorganism populations can also be affected by root exudates. The population and diversity of microorganisms were thought to be influenced by exudates released by plant roots24. Badri et al. [25] reported that the root exudates affect the formation of populations of rhizosphere microorganisms. According to Subba Rao [25], the variation of exudates released by the roots either directly or indirectly affects the quality and quantity of microorganisms in the roots. The content of organic compounds contained in it includes vitamins, enzymes to mineralization processes for the activity of microorganisms in cycles of nutrient 26, sugars, ethylene, polysaccharides, amino and organic acids, which trigger the development of microorganisms attracted by these compounds, especially in the plant's rhizosphere 27. Guckert et al. [25] reported that one of the factors that influence root exudate production is plant age and plant phase. The age of the Gondang manis Jamaica apple (Syzygium malaccense L), which reaches an average of 25 years, may affect the variety and distribution of exudate that comes out of the plant roots that grow and take firm root in the soil. Meanwhile, the plant canopy is tall with dense and thick leaves, with a height of up to 15 meters above the ground, causing the plant's environmental conditions to become humid. The older the plant is the more it supports the stability of the rhizosphere habitat around the environment. The results of research on groundwater in the area were also declared feasible as a source of irrigation 28 . In addition, in cultivation management, it generally uses organic fertilizers which is a factor in the existence of S. lanosoniveum as one of the fungi found in the area. Several studies related to S. lanosoniveum as a potential fungal biological control agent have been carried out, although based on the report of the fungi have not been widely used as a biogen, as is the case with Beauveria, Metarhizium, and Lecanicillium 16. The identification of Simplicillium lanosoniveum strain CG888 (MT 081944.1) as one of the biological agents among other fungi that have been found and identified can support the potential of suppressive soils in the Brantas watershed area. CONCLUSION Simplicillium lanosoniveum strain CG888 (MT 081944.1) is the identification of a fungus isolated from the exploration of the rhizosphere soil on Gondang Manis Jamaica apple (Syzygium malaccense L), Perak District, Jombang Regency, Indonesia, which is included in the Brantas watershed area. This is one of the antagonist fungi, so it can support the soil in the Brantas watershed as a potential soil suppressor. Therefore, to determine Simplicillium lanosoniveum strain CG888 (MT 081944.1) as an entomopathogenic and antagonistic fungus, it is necessary to carry out further testing of its pathogenicity against insects and pathogens that have potential as pests and diseases in cultivated plants in tropical climates. 020032-5 ACKNOWLEDGMENTS We would like to thank the President of the University of KH.A. Wahab Hasbullah and the Surabaya Plantation Seed and Plant Protection Center for their contribution so that research can be carried out properly. 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