Quantitative analysis of rhizosphere microbial population obtained from main-croplands in South Sulawesi

The study aims to investigate microbial species and quantify population richness associated with main crop rhizosphere in South Sulawesi. Survey was conducted in two areas including healthy and infected croplands. In each area about 1 ha, samples were collected to consist of 250 g bulk soil of rhizosphere in healthy and infected farms 1 month after transplanting. For analysis, those samples were carried out at the Plant pathology Lab, Department Plant Pest and Disease, Faculty of Agriculture. 10 g bulk soil separation out of 250 g obtained from rhizosphere was sterilized into water 25 mL and sieved 50-100 mesh prior to transfer to growth medium. After that, 4 % (g/mL) potato dextrose agar (PDA) in the petri dish was earlier prepared. The soil mixture (1g/10 mL) was diluted and transferred into a series dilution and parallelly loaded into solid media kept in dark room with at room temperature. Microbiota successfully growing in the solid media were split and transferred into petridish contained PDA for morphological identification and quantified through a series dilution. The study found 23 native fungal isolates consisting of Genera: Rhizopus sp. Phytophthora sp., Fusarium sp., Rhizoctonia sp., Geotrichum, Aspergillus sp., Gliocladium sp., Gongronella sp., and 14 unknown groups of fungi. Three bacterial isolates were discovered as gram-positive bacteria (GPB) in the infected cropland and the other two were gram-negative bacteria (GNB) found in healthy farm. The finding evidenced that population of microbiota ‘bacterial and fungal’ richness was twofold much higher in the healthy farm than infected farm. Bacterial population was the highest in both healthy and infected farms. There is a tendency of the higher fungal population richness, the lower rice disease incidence in croplands, vice versa.


Introduction
Environment, no exception to farmlands, nowadays has experienced a triangle-planetary issue like biodiversity losses as a forward and reverse consequence of multiple unsustainable practices [1].This manifestation threatens microbial communities and also depletes beneficial soil-nutrients [2,3].
Living microflora occupying plant rhizosphere zone are enormous and the role of their diversity in environment is important [2,3,4].In this area, it is a complex pathway that the root-host exudates encounter soil-borne microbes in many ways and share benefits vice versa [5,6].Once plant experiences due to pressing biotic nature, plant has resilient performance since root system generates H+ efflux and organic anions and secondary metabolites in the root exudates [6].Furthermore, in the context of beneficial microbes, the effect of microbial community within root system is able to combat primary crop-pathogen and pest directly in different way [7,8], generating crop protection services.A very convincing study demonstrated [9] that the way a tiny fungus generates a mode of action to kill a nematode whose the rest of life at most as a soil-borne pathogen in rhizosphere.Another indirect role is that their community activity is able to promote plant growth and enhanced nutrient availability [10] and nitrogen-fixation modules [11].They activate induced systemic resistance (ISR) of host against with suppressive pathogens living in the rhizosphere [4].
Regarding with plant immune mechanism, the rise of plant defense responses is generally elicited by response of plant pathogens.For instance, necrotrophic microbial pathogens induce plant immune through signaling pathways of jasmonic acid (JA), Ethylene (ET) and Reactive oxygen species (ROS) [12], and Hypersensitive reaction (HR) [13].Meanwhile, typical biothropic microbial plant pathogens induce salicylate acid (SA) signaling pathways [14].About 80% of rhizosphere microbes are able to synthesis and produce secondary metabolites that facilitate to overcome pathogen suppressions [15,16,17].
Last, microbiomes play important role of decomposing organic matters that enhanced soil nutrient [18,19] and increase of yield resilient.Microbes supply up to 30% of major nutrients needed for plant health such as phosphate and nitrogen.Conversely, excessive chemical substance apply evidenced to threaten their community population and poison living nature as well as environmental concerns [1].It evidenced that a prominent white filamentous fungus has a double-edged protection service, not just against from the major insect pest but also plant drought mitigation in marginal areas [8].
Many attempts were proposed to overcome the sources of biodiversity loss, but most are likely impractical way and cost effective since chemical substances use in environment is still most excessive [20].Optimizing microbes, therefore, associated with root system is put forward as a long-term encountered relationship in environment.This current study will investigate rhizosphere microbial species and population in both infected and healthy main crop-farms and analyze its diverse impact to food system.

Sample collection
The study focused on rice and corn farms which consisted of three mainly activities; namely sample collection, isolation and identification and quantification and data analysis.Collecting bulk soil method was followed by method of Braj Singh (pers.comm) where was conducted in main rice farm in Bulukumba district South Sulawesi which pose rice blast disease and health farm in 2021.Every farm (about 1 ha) diagonally and purposely chosen consisted of 5 blocks and collecting bulk soil was 200 g bulk soil obtained from 20 cm depth around rhizosphere and out of 200 g, 10g of interest was collected gently and poured into plastic bag transparent.Microbial isolation and morphological identification were conducted after they were grown onto potato dextrose agar (PDA) and Nutrient agar (NA).

Microbial characterization
A series of dilution method is a common technique to detect microbes associated with bulk soil.10 g bulk soil was diluted into 90 ml sterilized water and 10 mL obtained from first series dilution was transferred into another 90 mL sterilized water until sixth dilution.Quantification of microbes according to protocol of Braj Singh is that every 10 g bulk soil was determined with colony forming unit (CFU) which was representative of a total colony multiplying (x) with dilution factor (CFU/mL).fungal detection was matrix.In terms of bacterial diseases of plants diagnosis, the assessment was only determined to whether bacterial plant pathogenic role or not.The activity was divided into two namely morphological and physiological checks at least.Morphologically, bacterial colony was determined by using morphological matrix with parameters: colour, structure, form, elevation and margin and physiologically, the colony responded to chemical substance given such as (i) gram reaction of 3% KOH gently poured into glass-slide, (ii) the catalase enzyme test using 3% hydrogen peroxide (H2O2) to generate O2 and water (rapid bubble formation as indication of the presence of catalase), and (iii) hypersensitive response (HR) test to evaluate its pathogenic role (Figure 2).Microbial characterization was macroscopically undertaken.Rice disease incidence was generally analysed through the presence of symptom and disease incidence as follows:  HR test was predominant protocol to define bacterium of interest as pathogen or non-pathogenic role in the pathosystem [12].The indication was that the rise of lesion on tobacco leaf surface after artificial wound was meant by positive (+) reaction due to infection of plant pathogenic and by negative (-) or asymptom sign meant by non-plant pathogenic microbe.Non-plant pathogenic bacterium is well-known as 'a bacterial symbiont' in the plant rhizosphere having a mutual association with plant root system [10,11].In this current study, detection of bacterial role occupying plant rhizosphere was ensured by biochemical test.That is, gram test is a sufficient technique to examine its role in environment [21].Testing gram-negative bacterium (GNB) indicates as a role of bacterial plant pathogenic lifestyle while gram-positive bacterium (GPB) is defined as a common non-plant pathogenic bacteria as a role.

Microbial measurement
Growing microbes onto liquid media was undertaken prior to being transferred onto a series of microbial dilution technique such as first series of 1 mL diluted microbes load onto 9 mL sterilized water and subsequently a second series of 1 mL diluted obtained from first series dilution mixed with 9 mL sterilized water until forth diluted series. 1 mL as a final dilution was transferred onto solid media and counted their colonies following total plate count (TPC) protocol.One colony formed onto media represented to one microbial population.Colony forming unit was measured as follows:

Result and discussion
The average of microbial community in the three farm sites representative South Sulawesi has shown significant different higher population in the healthy farm than the farm with rice blast disease symptom according to Student T-test (P<0.05).Population of microbial rhizosphere community in croplands represented by every 10 g bulk soil was obtained in different location.Two main microbes are dominant, but bacterial and fungal population are significantly higher in healthy farm than infected cropland according to T test with P <00.5.In the cropland, the findings demonstrated that tremendous population fungi inhabited croplands and most likely as potential biological control agent.Morphological and physiological variation of bacterial group of interest in healthy and infected farms were discovered to associate with plant root system.In this finding, there were gram-negative bacterial pathogen population inhabited at most infected farm with a rice blast disease sign.This was evidence that, the response of bacterial disease to plant-host had a specific relationship as seen the HR test manifested as tobacco-leaf necrotic lesion (arrowhead; Figure 3).Plant tissue responded to infection strategy or early response of host to infection.In this case, a better understanding bacterial pathogenesis in the pathosystem is clearly demonstrated [21].It was stated that a gram-negative bacterial pathogen during early and late infection process promotes plant defense of programmed cell death (PCD) particularly a virulent bacterial type III secretion system.The PCD is well also known as hypersensitive response (HR) in the plant immune system.Majority of fungal population richness has inhabited rhizosphere in farm of Tambangan village with about 57% of total population.The least was about 4% proportion of fungal community in Tobirang Village (Figure 5).Although dominant fungal population richness was discovered in the Tambangan farm, disease incidence was quite high about 40% at the same area (Figure 4; Tabel 4).It evidenced that the role of encountered soil-borne microbiota population richness and plant root system is dynamic and complex [22].In this study, understanding the role of microbiota during interaction in environment is still partly unclear whether as a role of fully symbiont helping host resilient to disease or not.Yet, the finding has shown that there is a tendency of a light disease incidence occurrence in cropland once microbiota population richness is high (Figure 4).Oomycete Phytophthora sp.
Overall, the study found mainly 23 native fungal isolates in different Genera including Rhizopus sp., Oomycete Phythopthora sp., Fusarium sp., Rhizoctonia sp., Geotrichum sp., Aspergillus sp., Gliocladium sp. and Gongronella sp.Out of all findings, there are known Genera of plant pathogen such as Oomycete Phytophthora, Rhizoctonia and Fusarium in the pathosystem.The others were 14 unknown fungal isolates.The finding suggested that fungal population occupying plant roots in cropland system in Bulukumba district was significantly high.there is a tendence that the higher population richness, the lower RDI in croplands.However, in depth association between pathogen causing RDI and fungal population richness living in the plant-roots in different location is still yet to be fully unknow.

Figure 1 .
Figure 1.Sample "bulk soil" collection in healthy and infected plant roots

Figure 2 .
Figure 2. Typical single bacterial colony of interest on the NA-growth medium regarding with shape, elevation and margin appearance respectively.(a) punctiform; lobate; and convex and (b) circular; entire; and convex

Figure 3 .
Figure 3. Hypersensitive response (HR) test onto tobacco leaf surface after being 24-hours artificially injected with bacteria of interest.left: Lesion present (arrowhead).right: Lesion abstained (arrowhead).Host-tobacco leaf response to bacterial plant pathogen was used as a prominent protocol.

Figure 5 .
Figure 5. Proportion of fungal population richness inhabited different farms varied

Table 4 .
Rice disease incidence (RDI) and Genera of fungal microbiota and macroscopic characters in different farm in Bulukumba district-South Sulawesi.

Table 2 .
Testing morphological (m) and physiological (p) characters of bacterial colony of interest

Table 3 .
The illustration of micro-and macroscopic characters of overall fungal isolates obtained from 10 g bulk soil in different location