Organic rice rhizosphere fungi produce cellulase and chitinase as a biological control agent

Food crop security and security is a challenge in the agricultural sector to produce quality and healthy food products for consumption by the community. The use of synthetic fungicides to induce plant resistance negatively affects the abundance and diversity of biological agents.Cellulose enzymes and chitinase enzymes are capable lytic enzymes that degrade the cell walls of pathogenic fungi in plants. This study aims to isolate organic rice rhizosphere fungi and test their ability to produce cellulase and chitinase enzymes. Isolate the rice rhizosphere fungi using the spread-plate method, while testing the isolates producing cellulose enzymes using CMC mediaand testing isolates producing chitinase enzymes usingCDYA mediaadded with colloidal chitin.Penicillium sp. had the highest enzyme Hydrolysis Capacity (HC) value of 1.70 mm for cellulase and 1.19 mm for chitinase, followed byPaecilomycessp. with an HC of 1.52 mm for cellulaseand 1.12 mm for chitinase.Aspergillus sp. the HC of the cellulase and chitinase was 1.10 mm and 1.04 mm, while Trichoderma sp. the cellulose and the chitinase HC were 1.03 mm and 1.02 mm.


Introduction
The need for food supplies continues to increase globally, Food and Agriculture Organization (FAO) estimates that global food production from the agricultural sector needs to be increased by 70% to meet the food needs of the world's population in 2050 [1].Plants provide more than 80% of the food source and are the main source of nutrients consumed by humans.Yield losses in the global agricultural sector due to attacks by plant pathogens range up to 30% with an estimated global economic cost of up to hundreds of billions of dollars [2]. The increasing intensity of outbreaks of plant pests and diseases endangers the world's food and agricultural security systems.The use of chemical pesticides in the agricultural sector to prevent or control plant pathogens and weeds as well as efforts to reduce yield losses and maintain product qualityturned out to hurt biodiversity, the environment, also negatively impacts the health of agricultural workers and consumers and encourages the development of antimicrobial resistance [2,3,4,5].The international community lacks the capacity and coordination to prevent, eradicate and control plant pests and diseases, so it is important to have integrated pathogen management to reduce agricultural yield losses [5].
In the process of increasing crop production, there are herbicides, insecticides, fungicides, nematicides, fertilizers, and soil amendments currently used in high quantities.The chemicals have been used since the introduction of synthetic insecticides in the 1940s when Organochlorine Insecticides (OCl) were first used for pest control.Pesticides that reach the soil can change the diversity and microbial biomass of soil microflora thereby disrupting ecosystems and losing soil fertility and can ultimately affect plant growth [6].
Plant pathogens are difficult to control because their populations vary in genotype, space, and time [7].Control using biological agents is an alternative that effectively suppresses the growth and development of plant pathogens and does not cause negative impacts on the environment and the safety of food crops is maintained with the aim of a sustainable agricultural system [8,9].Rhizosphericmicroorganisms isolated from various plants produce different antagonistic compounds and inhibit the growth of various phytopathogenic microorganisms [9].
Most plant metabolites are released from the roots into the surrounding soil thereby triggering the selective growth of microbial communities so that microbial abundance is usually higher in the rhizosphere soil than in the surrounding bulk soil.Interaction between plants and rhizosphere microbes can affect growth, nutrition, development, disease susceptibility, contained nutrients, heavy metals, and xenobiotics.The soil media adjacent to the roots is called the ectorhizosphere which is colonized by mycorrhizal fungi.There is potential to engineer useful rhizosphere microorganisms, especially to increase the production of sustainable food crops [10].Rhizospheric fungi that are beneficial to plants are called Plant Growth Promoting Fungi (PGPF), they are very important in sustainable agriculture.GPPF can be used to reduce the use of agrochemicals in the management of plant diseases.PPGF produces defense enzymes, antibiotics, defensive/volatile compounds, and phytohormones thereby inducing resistance and long-term resistance in plants against pathogens [11].
The mechanism of antagonism of biological agents is direct antagonism including predation and hyperparasitism, indirect antagonism includes induction of host resistance and competition while mixed antagonism includes antibiotics and lytic enzymes namely protease, glucanase and chitinase which are capable of causing chemical and physical disruption of pathogens [12].The cellulose consists of three types of enzyme complexes including endoglucanase (endo-1,4--glucanase), cellobiohydrolases (exo-1,4--glucanase), and -glucosides, the three constituents of the cellulase work synergistically to convert complex carbohydrates present in lignocellulosic biomass into glucose, then this simple glucose is utilized by microorganisms as an energy source [13,14].
Chitinase is composed of structural polysaccharides -1,4-N-acetyl-D-glucosamine, chitin is one of the most common biodegradable polymers after cellulose [15,16].Chitin is abundant in the shells of crustaceans, insect exoskeletons, and some fungi.The chitinase is capable of degrading chitin in the cell walls of fungi and insect exoskeletons.Microorganisms that produce chitinase can be used as potential agents for the biological control of plant diseases caused by various insect pests and pathogenic fungi so that they can be used as an alternative to chemical pesticides [17].
Indonesia is an agricultural country with a tropical climate, which allows continuous cultivation in the agricultural sector with the consequent accumulation of pathogenic inoculums on agricultural land.Important biological controls are considered to be applied to reduce disease development and crop loss in different crop systems.The ability of rhizosphere fungi isolates to produce lytic enzymes has the potential as a biological agent against plant pathogens.This research is important to know the ability of organic rice rhizosphere fungi to produce cellulose and chitinase.

A sampling of organic rice rhizospheric soil
A sampling of the organic rice rhizosphere was carried out diagonally, which consisted of five sampling points on the plantation (figure 1).The rhizosphere samples taken were near the roots of the rice with a depth of ± 20 cm from the soil surface, then the rhizosphere samples at each sampling point were homogenized and then put in the sample envelope.

Isolation and purification of organic rice rhizospheric fungi
Isolation of rhizosphere fungi using the spread-plate method.Serial dilution of the rhizosphere samples was carried out to obtain a dilution of 10 -4 .A total of 1 g of sample was put into a test tube containing 9 ml of sterile distilled water and homogenized then taken 1 ml using an Eppendorf pipette then put into a test tube containing 9 ml of sterile distilled water after labeled with code 10 -1 .The results of each dilution are taken as much as 1 ml, put evenly into a cup containing Potato Dextrose Agar (PDA) media, and then incubated for 3-7 days at 30℃.After the incubation period, isolates of fungi with different morphological characteristics were purified ina new PDA medium [18].

Identification of organic rice rhizospheric fungi
Observing macroscopic and microscopic morphological characters using a 40x magnification binocular microscope.The observations included the color and surface of the colony, radial lines from the center towards the edge of the colony, concentric circles, hyphae structure, and reproductive structures (presence or absence of rhizoids, conidia, and spores).The identification of the fungus refers to the Illustrated Genera of Imperfect identification bookFungi [19].

Activity test of cellulose and chitinase of organic rice rhizospheric fungi
Testing of isolates producing cellulose using Carboxymethyl Cellulose (CMC) selective agar, containing 0.2% NaNO3, 0.1% K2HPO4, 0.05% KCl, 0.2% sodium salt, 0.02% peptone, 1.7% Difco Agar, and Remazol Bronthimol Blue (RBB).Pure isolates of rice rhizosphere fungi were grown on CMC media and then incubated at 30℃.After 72 hours of incubation, the cup containing the fungus was poured with gram iodine solution for 3-5 minutes after which the gram iodine solution was removed from the petri dish [20].Furthermore, testing the isolates producing the chitinase by making Czapek Dox Yeast Agar (CDYA) media added 0.1 g of dried shrimp shell powderand 0.2% sodium salt then the rhizosphere fungi isolates were grown on the media, then incubated at 37℃ for 3 days [21].
Calculation of the hydrolysis capacity index measured on the third day of observation will show a clear zone on the planting medium intended for each enzyme tested so that the ability level of each fungus culture to hydrolyze substrates is known.The following is the hydrolysis index calculation formula capacity [20].

Data Analysis
The type of research used was descriptive quantitative research by conducting research directly to obtain the results of isolating organic rice rhizosphere fungi and testing cellulose enzymes and chitinase enzymes to obtain the hydrolysis capacity index of the enzymes tested.The data obtained are presented in the form of pictures and quantitative data is then processed descriptively.

Identification of organic rice rhizospheric fungus isolation results
The results of the isolation of organic rice rhizosphere fungi obtained 4 isolates from the genus of fungi namely Trichoderma sp., Paecilomyces sp.,Penicillium sp., and Aspergillus sp.Trichoderma sp. has radial colonial growth with a clear ring pattern, green and white colonies and the base of the colony is whitish, flat surface is round in shape but rough texture like fibrous, thick fine mycelium has a velvety texture, and the conidia are round and grow at the ends of conidiophores, conidiophores branch like pyramids i.e. the branches are longer underneath, the phialides stalks are short and arranged in different groups, there are 2-3 fields per group (figure 2 RF1).Conidiophore of Trichoderma sp.hyaline colored, branched, single phialide on each branch, conidia globose and have fast growth [19].
Paecilomycessp.has a thick mycelium and forms conidiophores.There are phialides at the end of the spores that form in long chains.Spores will germinate when moisture and nutrients are available.Vegetative hyphae are smooth walled, and hyaline.The phialides are swollen at the basal part and taper to the neck.Conidia are unicellular and in chains, in different chains, they are fusiform ellipsoid, oval, and smoothwalled(figure 2 RF2) [22].Aspergillus sp. has bright green colonies and the base of the colony is yellowish white, a fine and thin mycelium with a texture like cotton and flat colony edges visible.The spores are clearly visible on the surface of the radial colonies, the conidia are round, the vesicles are round and the conidiophores are long and cylindrical, and the colonies and spores are green with white colony edges visible (figure 2 FR3).Aspergillus sp. has green colonies with white colony edges, has a texture mycelium is velvety or woolly with a flocosal center, conidiophores are colorless, thick-walled, rough, and have cushioned vesicles, vesicles are spherical to sub-globose, metula cover the surface of the vesicles and are located in all directions, conidia are round, thin-walled and slightly rough [22,23].
Penicillium sp. have grayish-green colonies with white edges, the base of the colonies is yellow, the colonies are oval with serrated edges and a dense texture, the conidia are round at the end of the conidiophore series, the conidiophores are upright and branched with a collection of cylindrical phialide at the end (figure 2 RF4).Penicillium sp. has conidiophores upright and circularly branched either single or multiple, and resembles a bush-like branching.Conidia are produced at the end of the series, the shape is round, the conidiophores are finger and there are 2-3 branching hyphae [19].

Hydrolysis activity test of cellulase and chitinase of organic rice rhizospheric fungi
The ability of rhizosphere fungi isolates to secrete cellulase was indicated by the presence of a clear zone on the CMCmedia (figure3).The ability of isolates of rhizosphere fungi to produce the chitinase index was indicated by the presence of clear zones on the CDYA media with colloidal chitinusing shrimp shell powder (figure 4).The ability of rhizosphere fungi to produce antibiotics is very important in determining the ability of colonization and the extracellular lytic enzymes produced can cause endolysis or autolysis so that pathogens experience inhibition or death.The following provides a breakdown of the hydrolysis capacity index of the chitinase enzyme by each rhizosphere fungus isolate.Based on the clear zone formed due to the hydrolysis activity of rice rhizosphere fungi in the planting medium which is intended to detect the ability to produce the enzymes tested, it was obtained highest ratio value is the isolate Penicillium sp.formed 1.7 mm cellulose HC and 1.19 mm chitinase HC then 1.52 mm cellulose HC and 1.12 mm chitinase HC in Paecilomyces sp.isolates.The lowest HC ratio was found in Trichoderma sp.formed 1.03 mm cellulose HC and 1.02 mm chitinase HC while Aspergillus sp.formed 1.1 mm cellulose HC and 1.04 mm chitinase HC (table 1).
The presence of extracellular enzymes based on the test results, the mechanism of antagonism of Trichoderma sp.against pathogenic fungi is carried out by releasing toxins in the form of chitinase, β-1,3glucanase and cellulose thereby degrading the cell walls of pathogens [14,24].Paecilomycessp.can live in various habitats such as soil, forest, grass, desert, and silt so that its distribution is very wide.Based on the phylogenetic tree analysis, it is known that Paecilomycessp.as a producer of extracellular lytic enzymes, namely cellulase, and chitinase which function as chitinolytic, can degrade the cell walls of fungal and insect pathogens [25,26].
Aspergillus sp.produces cellulose and can produce chitinase which is catalytic so that they are useful for the biodegradation of chitin end products and biocontrol of organisms, shrimp is a source of substrate that can be utilized by Aspergillus sp. to produce chitinase [14,21].Penicillium sp. is capable of producing cellulolytic (β-exoglucanase, β-endoglucanase, and β-glucosidase), and is also capable of producing chitinase.This statement proves that Penicillium sp. has the potential as a biological agent that can be used in the agricultural sector as an integrated control of plant pathogens [13,14,26,27].
Trichoderma sp. and Paecilomyces sp.besides having the ability to produce lytic enzymes, it is also a mycoparasite of pathogens to inhibit or stop the growth and development of plant pathogens.Mycoparasites are fungi that parasitize other fungi.The parasitic properties of fungi are grouped into two categories, namely biotrophic and necrotrophic mycoparasites.In biotrophic mycoparasitic fungi form specialized feeding relationships, usually by producing haustoria to penetrate and absorb nutrients from living fungal hyphae.In contrast, in necrotrophic mycoparasitic, the fungus attacks and destroys other fungal cells and feeds on the contents of the dead cells [28].Aspergillus sp. and Penicillium sp. as biological agents through the mechanism of antibiosis.Biological agents that have an antagonistic mechanism, namely antibiosis, are biological agents capable of producing antibiotics and lytic enzymes, namely protease, glucanase, cellulose, and chitinase, which are capable of causing chemical and physical disruption of pathogens [12].

Conclusion
There were 2 isolates of organic rice rhizosphere fungi that had the highest Hydrolysis Capacity (HC) index in the chitinase and cellulase tests, namely Penicillium sp. a cellulase HC index of 1.70 mm and a chitinase HC index of 1.19 mm, as well as Paecilomyces sp. the cellulase HC index was 1.52 mm and the chitinase HC index was 1.12 mm.

Table 1 .
The ability of organic rice rhizosphere fungi to produce cellulase and chitinase is based on the inhibition zones produced.