Exploration and macroscopic characterization of microbiomes (fungi and bacteria) on anthracnose-Infected chili plants in lowlands fields of Deli Serdang North Sumatra

Anthracnose is one of the main obstacles in chili cultivation. This disease reduces chili production by 40% to 100% during the rainy season. Most techniques used to control anthracnose is using synthetic fungicides that potentially cause resistance and environmental pollution. Disease management by the microbiome of plants with antagonistic properties is an alternative biological control. The aim of the study was to explore the diversity of the microbiome (fungi and bacteria) in various tissues (rhizosphere, endosphere and phylosphere) of large red chili plants infected with anthracnose macroscopically. The object of this research was the leaves, stems, and roots of 5 months old of chili plants affected by anthracnose from two different locations of planting areas. The exploration of the microbiome of anthracnose- infected chili plants in a lowland area in Sumatera Utara in 3 DAI discovered 13 fungi and 12 bacteria, whilst 10 fungi and 11 bacteria in the second location by tissue cropping technique. Besides that, by grinding technique, resulted in 14 fungi isolates dan 13 bacteria in the first location and 16 fungi isolates and 14 bacteria in the second location. At 7 DAI, there found 130 bacteria in the first location and 146 bacteria in location 2 by grinding technique. By tissue cropping technique, there were 124 bacteria in the first location and 102 bacteria in the second location. Morphologically, fungi were identified as Fusariumsp, Aspergillus sp, Penicillum sp, Trichoderma sp, Humicola sp, Colleotetrichum sp, Idriella sp, Alternaria sp, Rhizopus sp and Curvularia sp while the bacteria obtained 9 different isolates based on the morphology of colony.


Introduction
Chili is a horticultural crop that has high economic value and contributes to the production of seasonal vegetable commodities in Indonesia, especially in North Sumatra.This is because chili is needed by Indonesian people as a spice in daily cooking, food raw materials, and pharmaceuticals [1][2][3][4].The red chili production in Indonesia in 2021 was 1.36 million tons and North Sumatra contributed 15.46 percent (210.22 thousand tons) [5].In Indonesia, the increase of needed for chili reaches 0.5-1% per year but this is not in line with the resulting production.
The decrease of chili production can be caused by pests and diseases.The anthracnose caused by Colletotrichum spp. is one of the most harmful diseases.Anthracnose attacks the stem, leaves, and fruits.The symptoms of anthracnose disease start from the lower part of the plant.The leaves, twigs, and branches become dry, blackish brown, the fruit shrinks and dry with a black colour.According to [6] the fungus Colletotrichum spp can be carried by seeds and it will attack plants in nurseries and in the field.The fungi can survive on plant residues or diseased fruit, and it can be a source of transmission.Those environmental factors intensify the disease attacks and wider the disease spread.Yield loss on production chili due to anthracnose attacks can reach 50-100% in the rainy season [7] and 20 -35% in dry season.To control the disperse of the disease, farmers use high doses of synthetic fungicide with high intensity.This can affect the environment because of the pollution and the residues causing the resistance and decrease the plant microbiomes.The plant microbiome includes the microbial community interacts extensively with plants.The plant microbiome can survive both inside and outside of plant tissues, carrying out a variety of activities that are beneficial to plants.
These few years, alternative disease control by exploring microbial resources found in the rhizosphere, endosphere and philosphere (bacteria, archaea, viruses, fungi, and eukaryotic microorganisms) of plants [8].The microbiome from plants is called the plant microbiome.According to Vorholt (2012) [9], plant microbiomes is microorganism collected from rhizosphere and philosphere area (which are near or on plant tissues) and it was considered epiphy.microbial in plant tissues (endosphere), whether in leaves, roots, or stems, are considered endophytes [10].
The microbiome community is influenced by four factors, geographic location, soil sources, host genotype, and cultivation practices.Based on the factors, then do study is carried out including isolation of microbes, visualization using macroscopic and microscopic characters, biochemical testing on bacteria, secondary metabolic analysis and metabarcoding of functional genes to analyze microbial meta-transcriptomic, metaproteomic, and metabolomic activity.[11].
Based on this, the development of the microbiome as an anthracnose disease biocontrol agent in the field into environmentally friendly products has a great opportunity to reduce the incidence of plant diseases and reduce chemical inputs [12].The aim of the study was to discover the diversity of culturable the microbiome (fungi and bacteria) in various tissues (the rhizosphere, endosphere, and phylosphere) of red chili plants infected by anthracnose.

Research Methodology 2.1. Laboratory experiments
This research was conducted in the Plant Pathology Laboratory at the University of Sumatera Utara.The research was from February to March 2023.The materials used rhizosphere, endosphere, and phylosphere tissues of Local red chili infected by anthracnose in the lowland fields.The equipment used were petri dishes, erlenmeyer, test tube, microscope slides, autoclave, oven, Laminar Air Flow Cabinet (LAFC), microscopes, needles, stationery, and camera.The samples were 5 months old with 50% anthracnose infection.The selected location was former paddy fields planted with chili 0,5 ha wide.Samples explored for their microbes including root, stem, and leaf tissues.The surface of the samples was sterilized using distilled water twice for 1 minute [13].For microbial exploration, this study used 2 techniques (tissue planting and grinding technique).
The planting technique was carried out by cleaning the surface of the sample roots, stems, and leaves which had been cut to a size of 1 x 1 cm and then sown on agar media PDA and NA.In the grinding technique, the washed plant parts were cut into samples of 10 g which has been cleaned is then processed using a mortar dan pestle.The sample extract was diluted using distilled water 8 times to achieve the proper microbiome density (10 -7 ).0.1 ml dilution piped into a petri dish containing PDA and NA using a micropipette.The suspension is flattened with the L stick until the suspension is evenly distributed in the media and incubated for 7 days for observation.
Fungal purification possesses different steps regarding their macroscopic appearance, for instance the colony shape color.The microbes identified and separated between bacteria and fungi.For bacteria developed in NA media and fungi developed in PDA media.The zig-zag streak method was applied for bacterial purification.The identification of bacteria was carried out based on macroscopic appearance.

Variables
The variables observed include the number of fungi and bacteria from exploring the microbiome in rhizosphere, endosphere, and phylosphere.The observed characters were hyphae and conidia.Further identification was carried out under a microscope and matched with the reference book Pictorial Atlas of Soil and Seed Fungi.The classification of the bacteria includes colony form, elevation, edge shape, and colony color.

Microbiome exploration (fungi and bacteria)
The exploration of the microbiome (fungi and bacteria) of chili plants infected with anthracnose in lowland rice fields found several potential microbiomes.Based on planting techniques and grinding techniques on PDA Media with a cropping system presented in Table 1 and Table 2 1 showed the microbiome successfully explored at location 1 and location 2. Three day after inoculation (DAI), the first location inquire 13 fungi isolates and 12 different bacterial isolates from roots, stems, and leaves (Figure 1).At the second location, 10 fungi isolates, and 11 different bacterial isolates appear on the growth medium (Figure 2).A further result showed that in the 7 DAI, there were 124 bacteria (57 from root, 42 from steam, and 25 from leaves) discovered in the first location.For the second location, 102 bacteria colony (43 from root, 30 from steam, and 29 from leaves) appears from the growth media.The difference in the number of microbes explored is due to differences in cultivation practices in the field while the chilies varieties used are the same.The composition of the microbiome varies based on soil sources, and genotypes.field conditions, geographical location, and cultivation practices, namely organic vs. conventional [14,15].Table 2 showed the microbiomes that were successfully explored from the technical results of grinding with the scattering system.At location 1 found 14 fungal isolates and 13 different bacterial isolates from roots, stems, and leaves while in the second location, 16 fungal isolates and 14 different bacterial isolates were found.At 7 DAI there were 130 bacteria isolation (57 from root, 43 from steam and 30 from leaves) from the first location and 146 bacteria (64 from root, 47 from steam and 35 from leaves) in the second location (Figure 3 and Figure 4).Table 2 shows that root tissue has more microbiomes in locations 1 and 2, followed by stem tissue.Based on the planting technique, it shows that the grinding (spreading) technique has a higher number of microbiomes (fungi and bacteria) than the planting tissue technique.

Fungi
The microbiome being explored is a fungus originating from the roots, stems, and leaves of red chili plants infected with anthracnose.At locations 1 and 2, fungi were obtained which were then given a code starting with the letters L1S1 and L2S1 followed by the network origin code and number.Macroscopic characters were observed 7 days after incubation (DAI).The characters observed were the pattern of mycelium distribution, the colour of the colonies above and below the surface of the media, the texture of the colonies, while for bacteria it was seen from the texture of the colonies.The macroscopic characters of the microbiome (fungi) isolates are presented in Table 3. Concentric White Green Aspergillus Table 3 showed the pattern of mycelium distribution, two different types of morphospecies were obtained, namely concentric and radial growth of fungus colonies.The fungal isolates grew radially (spreading from the center of the colony to the edges of the colony), while the other 26 isolates had mycelium that grew in a concentric pattern originating from the roots, stems, and leaves of chili plants infected with anthracnose.The colour of the microbiome colonies was found to be vary tended to be dominated by white.

Fungi Identification
Microscopic characterization of endophytic fungi was carried out using a 40x magnification optimal photomicroscope.The observed characters were hyphae and conidia.Further identification was carried out under a microscope and matched with the reference book Pictorial Atlas of Soil and Seed Fungi [15].Based on the results of microscopic characteristics.Identification results showed that there were 10 fungi that were successfully identified and 3 could not be identified because of the imperfect body of the fungi.The characteristics and identification of each isolate are described in Table 4.In Fusarium microscopic observations, the characteristics of conidiophores are branching, microconidia are ovoid in shape with 1 cell, hyaline, and smooth walls, macroconidia are cylindrically consisting of 2-3 cells, chlamydospores are not found, hyphae are insulating [16].

Number of Isolates
Microscopic Observations on isolates L1SBCJ3, L2SDCJ5, L2SBCJ1, L2SACJ5, L2SBCJ2 have hyaline-colored hyphae structures, hyaline-colored conidiophores, simple long straight shapes, slightly curved edges, subglobose conidia [17].The Aspergillus sp has conidia-shaped heads 4 adiate, round, there are phialids at the head of the conidia, the conidiophore is long and swells into a vesicle at the end carrying a sterigma where conidia grow, has aseptate hyphae [18] .Aspergillus sp. has asexual reproduction by producing spores called conidia.This isolate was identified as Aspergillus sp [19].
Isolates L1SACJ4, L1SACJ5, L1SBCJ1, L2SBCJ5 showed the fungus had hyaline-coloured hyphae, the conidiophores grew branching and formed several fialids at the ends.Conidia are hyaline in color and grow at the ends of the fialids.Conidia are round and form long chains.Penicillium sp. have conidiophores that arise from the mycelium.At the end of the conidiophore, it will branch out to form a group of fialids.Conidia are hyaline or brightly coloured.Most of the conidia are ovoid and form long chains [20] Isolates L1SDCJ5, L1SBACJ1, L2SBCJ3 showed that the fungus has upright conidiophores, many branches, short and thick fialids, conidia are round to ovoid in shape and are single-celled, somewhat conical in shape, can form chlamydospores, is the fungus Trichoderma sp.Trichoderma sp has conidiophores that are highly branched and upright.Conidia are ovoid in shape with short, thick fialids [21].The isolates L1SBCJ2 and L2SACJ4 were observed microscopically and showed that the fungi had round conidia and short and perpendicular conidiophore branches.the conidiophores are upright, have short branches, single apical conidia, 1-celled fialids tapering upwards to produce small, round conidia.Humicola belongs to the Sordariomycetes class, the order Sordariales, and the family Chaetomiaceae [22].
Microscopic observation of isolates L2SBCJ4 and L2SDCJ1 showed that the fungus has insulated and branched hyphae and produces conidia that are transparent and elongated with rounded or tapered ends, between 10-16 µm long and 5-7 µm wide with black conidial masses.The Colletotrichum gloeosporioides mushroom has a cylindrical spore shape, the spore tip is blunt, the spore size is 16.1 x 5.6 µm, while the Colletotrichum acutatum mushroom has a cylindrical spore shape, the spore tip is tapered, the spore size is 16.1 x 5.3 µm with a growth rate of 6.8 mm per day.The fungus Colletotrichum capsici has a spore shape like a crescent moon, the tip of the spore is pointed, the spore size is 24.3 x 4.4 µm.[23] The results of microscopic observations showed that Idriella sp. had short conidia in the form of the lunate (sickle) to elongated falcate with slightly curved tips, which were hyaline and unicellular.Chlamydospores in clavate mushrooms (enlarge at the ends or like clubs.Based on microscopic observations, isolate L1SACJ2 belongs to the fungus Idriella sp.
The fungal isolate L1SDCJ1 has dark conidiophore, rather short, dark conidia, usually both elongated septa of various shapes, oval to elliptical or ovoid.Fungus Alternaria spp has erect and short septa hyphae.Conidia show ovate or ellipsoidal shape, simple or branched, often with a small hilum.That Microscopic observation of isolates L1SACJ2, L2SACJ1 showed that the fungus Rhizopus spp., have rhizoids and stolons, oval-shaped sporangium and non-septate hyphae.

Bacteria
Bacterial characterization was carried out on all isolates found on the 3rd day after incubation.Bacterial characterization was carried out on all isolates found on the 3 rd DAI.All total isolates produced until day 7 had the same characteristics as the bacteria that had been tested.Bacterial isolates were given a code starting with the letters BC (chilli bacteria) followed by the tissue origin code and number.Characterization of bacteria includes colony morphology (shape, elevation, edge shape, surface, and colour of the colony), and cell morphology which includes cell shape (Table 5).

Conclusion
The exploration of the microbiome of anthracnose-infected chili plants in a lowland area in Sumatera Utara in 3 DAI discovered 13 fungi and 12 bacteria, whilst 10 fungi and 11 bacteria in the second location by tissue cropping technique.Besides that, by grinding technique, resulted 14 fungi isolates dan 13 bacteria in the first location and 16 fungi isolates and 14 bacteria in the second location.At 7 DAI, there found 130 bacteria in the first location and 146 bacteria in location 2 by grinding technique.By tissue cropping technique, there were 124 bacteria in the first location and 102 bacteria in the second location.Most of the microbiomes were found in root tissue followed by stems and leaves.In the fungi microbiome, 10 fungi were identified, Fusarium sp, Aspergillus sp, Penicillum sp, Trichoderma sp, Humicola sp, Colleotetrichum sp, Idriella sp, Alternaria sp, Rhizopus sp and Curvularia sp while the bacteria obtained 9 different isolates based on the morphology of colony.

2
Anthracnose infected chilli plants during the rainy season or wet climates with high rainfall.

Table 1 .
. Results of microbiome exploration of chili plants infected with anthracnose through tissue cropping technique on PDA media.

Table 2 .
Microbiome exploration results of chili plants infected with anthracnose by grinding technique on PDA media.

Table 3 .
Morphological characteristics of the microbiome (fungi) of red chili plant tissue macroscopically infected with microscope