Biocontrol Potential of Rhizosphere Fungi Against Pyricularia Oryzae in Ciherang Rice Variety

. Blast disease is an important disease of rice caused by Pyricularia oryzae. The use of antagonistic fungi from the rhizosphere of rice plants has the potential to control plant pathogens. One of them is from the genus Aspergillus. This research aims to test the ability of Aspergillus genus fungi in suppressing the intensity of P. oryzae attack on ciherang rice varieties. The research methods were isolation and identification of rhizosphere fungi, propagation of P. oryzae , antagonistic test of rhizosphere fungi against P. oryzae in vivo, and reisolation of fungi from rice plant tissues. This research used a completely randomised design (CRD) consisting of 9 treatments and 3 replications. Data obtained from this research were statistically analysed using analysis of variance (ANOVA) and Tukey's honest significant difference test at the 5% level The results showed that Aspergillus sp1 and Aspergillus sp2 fungi could significantly reduce the intensity of blast disease attack caused by P. oryzae isolates from Moncongloe and Simbang regions..


Introduction
Food demand continues to increase therefore to meet global food demand by 2050 agricultural production must be increased by 70% [1].Climate change will affect agricultural systems through higher temperatures, changes in rainfall, increased weeds, pest and disease pressure.Losses caused by pests and diseases are estimated to average 21.5% in wheat, 30.0% in rice, 22.6% in maize, 17.2% in potatoes, and 21.4% in soybeans [2].One of the causes of yield loss in rice is blast disease.Blast disease is caused by the pathogen Pyricularia oryzae [3].The reduction of rice yield in Japan due to blast disease was about 60%, in Brazil it reached 100%, India 7.5%, Korea 8%, China 14%, Philippines 67%, Vietnam 60%, Italy 24% and Iran 50%.In Indonesia, blast disease explosion can cause yield losses of about 50%-90% in susceptible rice plants [4].This pathogen is difficult to control because it has high genetic diversity [5] and excellent adaptability.Pathogen races have virulence properties that can change rapidly according to host and environmental conditions.[6] the results of research on blast disease intensity in rice plants in endemic areas were 55.60% for leaf blast, and 37.75% for panicle neck blast.The potential yield loss is estimated to be around 3.65 tonnes/ha or equivalent to 61% yield loss when compared to the average rice production according to variety specifications.Various types of control that can suppress P. oryzae include the use of synthetic fungicides, which are considered the most effective in controlling blast disease, although they have negative effects [7].Control using resistant varieties does not provide maximum results because resistant varieties can only last about two to three seasons.This is because the blast pathogen has the ability to adapt well and mutations easily occur in the genetic makeup of the P. oryzae pathogen.
One of the effective control alternatives is to use biological agents in the form of antagonistic fungi from rhizosper that can suppress the growth of the blast pathogen.Biological control by utilising biocontrol agents is known to induce plant resistance to plant pathogens and abiotic stresses [8], Endophytic fungi are fungi that can live in plant tissues for all parts of their life cycle without causing damage or disease to the host.Endophytic fungi isolated from IR 42 and Cisokan rice belong to the genus Aspergillus, Trichoderma and Beauveria [9].Aspergillus sp. and its metabolites can function as biocontrol agents against several plant pathogens can inhibit rice sheath blight fungus and can show strong antagonistic activity against Rhizoctonia solani.Cyclopyazonic acid production from Aspergillus sp strain R3 significantly reduced leaf blight severity in rice seedlings infected with R. solani.The fungus Aspergillus terreus has a bioactivity profile of the active molecule terrein that can act as an antibacterial, antifungal, and cytotoxic and fungal biocontrol.The biocontrol potential of microorganisms against leaf spot disease in ginger plants is helpful in minimising the use of agrochemicals and making agriculture more productive and sustainable, A.terreus is an endophytic soil fungus, which can promote plant growth and can show strong biocontrol activity, can inhibit the growth of P. oryzae by competing for space and nutrients and is also parasitisme [10] described the role of A. terreus as a plant growth promoter and biocontrol microorganism [11] The fungus A. terreus isolated from the soil in suppressing the pathogen Sclerotinia sp, the results obtained were that A. terreus was hyperparasitic and caused 100% death of S. sclerotium sclerotia.Aspergillus fumigatus showed significant antifungal activity against the coffee phytopathogens A. alternata and Fusarium incarnatum .The fungi A. flavus, A. fumigatus, A. niger inhibit by competing, lysing the pathogen cell wall, and producing antibiotic substances, which is called the antibiosis mechanism.In general, the fungi A. fumigatus and A. terreus can be used as pathogen antagonists.[12], Previous research informed that in the rhizosphere area of rice plants there are several fungi that have the potential to control Pyricularia oryzae infection, including A. flavus, A. fumigatus, A. niger, Curvularia sp. and Trichoderma harzianum [13].This research is very important to test the ability of antagonistic fungi from the rhizosphere of rice plants, especially the genus Aspergillus against P. oryzae isolates from different regions.Therefore, these fungi have the potential to be developed as candidate biocontrol agents against blast disease.

Isolation of rice rhizosphere fungi
Fungi from the rhizosphere of healthy rice plants were isolated using the scatter dish method.Serial dilutions were performed to obtain a 10 -4 dilution.Soil sample 1 g sample was put into a test tube containing 9 ml of sterile distilled water and homogenised then 1 ml was taken using an Eppendorf pipette and then put into a test tube containing 9 ml of sterile distilled water after being labelled 10 -1 .The result of the dilution was taken as much as 1 ml, flattened into a cup containing Potato Dextrose Agar (PDA) media, incubated for 3-7 days at 30℃.Separation of fungal isolates with different morphological characteristics was purified in new PDA media [14].

Identification of rice rhizosphere fungi
Pure fungal isolates were observed for macroscopic and microscopic morphological characters using a 40x magnification binocular microscope.Observations include the colour and surface of the colony, radial lines from the centre to the edge of the colony, concentric circles, hyphal structure, and reproduction (presence or absence of rhizoids, conidia, and spores).Fungal identification refers to the book Illustrated Genera of Imperfect Identification of Fungi [15].

Pathogenic fungus inoculum preparation
The pathogenic fungi used were isolates of P. oryzae fungi that had been isolated from 3 different areas in Maros Regency, namely (Mandai, Moncongloe, Simbang).Inoculum was prepared by multiplying pure culture of P.oryzae fungus on PDA media.Description: ( Po1) P. oryzae from Mandai region ; (Po2) P. oryzae from Moncongloe region; ( Po3) P oryzae from Simbang region.

Antagonistic test of rhizoper fungi against P.oryzae in vivo
Preparation of planting media is soil mixed with manure in a ratio of 2:1.After mixing well, the planting media was put into a plastic pot as a container for plants.The rice variety used as a test plant is the Ciherang variety.Each plant was inoculated with 2 ml of antagonistic fungus suspension first at 23 days after inoculation while the pathogen was applied at 30 dai with 2 ml of P. oryzae suspension.The suspension was sprayed on the underside of the leaves (stomatal area) of the rice plants.The  This research uses a completely randomised design (CRD).There were 9 treatments and 3 replications.Observations were made 7 days after inoculation based on the observation scale of the standard evaluation system for rice (IRRI) presented in Table 1.Spot type score 5-9 was used to identify plant susceptibility.The formula used in calculating disease intensity is as follows: Description I disease intensity (%), n number of leaves with a certain score, v score value of each leaf, N number of leaves observed, V The highest score of leaf blast attack.

Reisolasi of fungi from rice plant
Isolation was carried out by taking rice plant tissue, then surface sterilisation and then placed on PDA media and incubated for 2 weeks.The growing fungi were purified and re-identified to prove that antagonistic and pathogenic fungi still exist in rice plants.

Data analysis
The data obtained from this research were statistically analyzed using the analysis of variance (ANOVA).If the data is found to be significantly different, then it is continued with Tukey' Honestly Significance Difference (HSD) test at the 5% level

Results and discussion
The results of isolation and identification of rice rhizosphere fungi found two fungi that have different characteristics.Based on macroscopic and microscopic identification, these fungi belong to the genus Aspergillus, namely Aspergillus sp1 and Aspergillus sp2.The percentage of blast disease intensity caused by P. oryzae isolated from Mandai region after application of 2 antagonist fungi isolates (genus Aspergillus) is shown in Figure 1.    1, observations at 7 days after inoculation (dai) showed the highest intensity in the treatment of P. oryzae with Aspergillus sp2. was 5.26%, then followed by the treatment of P. oryzae with Aspergillus sp1. was 4.87% and the lowest intensity was seen in the control treatment was 4.73%.Observation of 9 dai showed an increase in disease intensity in each treatment.The highest intensity in the treatment of P. oryzae with Aspergillus sp2 was 6.84%, then in the treatment of P. oryzae with Aspergillus sp1 was 6.28% and the lowest intensity in the control treatment was 5.60%.However, at 11 dai, the highest disease intensity was seen in the control treatment P. oryzae was 7.70%, then the treatment of P. oryzae with Aspergillus sp2 which was 6.94%, and the lowest intensity was seen in the treatment of P. oryzae with Aspergillus sp1. was 6.76%.Observation at 13 dai, the highest intensity was seen in the control treatment was 8.93%, then the treatment of P. oryzae with Aspergillus sp1 was 7.56% and the lowest intensity was seen in the treatment of P. oryzae with Apergillus sp2 was 6.97%.Based on the table of leaf blast disease severity scale, data at 13 days observation can be determined that the control treatment shows the criteria of highly susceptible and the treatment of P. oryzae with Aspergillus sp2.and treatment of P. oryzae with Aspergillus sp1.shows the criteria of susceptible.
The percentage of blast disease intensity caused by P. oryzae isolated from Moncongloe region after application of 2 antagonist fungi isolates (genus Aspergillus) is shown in Figure 2. Figure 2, at 7 days observation showed the highest intensity is seen in the treatment of P. oryzae with Aspergillus sp2. was 4.67%, then followed by the treatment of P. oryzae with Aspergillus sp1. was 3.99% and the lowest intensity is seen in the control treatment was 3.76%.At 9 days , there was an increase in disease intensity in each treatment.The highest intensity was still seen in the treatment of P. oryzae with Aspergillus sp2. was 6.39%, then in the control treatment was 5.70% and the lowest intensity was shown in the treatment of P. oryzae with Aspergillus sp1. was 5.52%.However, at 11 days,the control treatment exhibited the highest intensity (7.67%), followed by the P. oryzae treatment with Aspergillus sp2.(6.63%) and the P. oryzae treatment with Aspergilus sp1 (5.98%).Observations at 13 days showed that the highest intensity was seen in the control treatment ( 9.00%) , then in the treatment of P. oryzae with Aspergillus sp2 was 7.00% and the lowest intensity in the treatment of P. oryzae with Aspergillus sp1. was 6.00%.The percentage of blast disease intensity caused by P. oryzae isolated from Simbang region after application of 2 antagonist fungi isolates (genus Aspergillus) is shown in Figure 3 .Figure 3, 7 days observation showed the highest intensity is shown in the control treatment was 5.88%, then followed by the treatment of P. oryzae with Aspergillus sp1. was 4,21% and the lowest intensity of P.oryzae with Aspergillus sp2 was 4,02%.At 9 days , there was an increase in disease intensity in each treatment.The highest intensity is shown in the control treatment at 7.35% then in the treatment of P. oryzae with Aspergillus sp1 at 6.05%, and the lowest intensity was seen in the treatment of P. oryzae with Aspergillus sp2. at 5.83%.At 11 days, the highest intensity of blast attack was in the control treatment at 8,00%, then in the treatment of P. oryzae with Aspergillus sp1 at 6.00%, and also the treatment of P. oryzae with Aspergillus sp2 at 6.00%.Observations at 13 days showed the highest intensity in the control treatment was 8.00%, then the treatment of P. oryzae with Aspergillus sp2. was 6.00% and the treatment of P. oryzae with Aspergillus sp1 was 6.00%.

Reisolation of fungi in rice
The isolation and identification results showed that Pyricularia oryzae, Aspergillus sp1 and Aspergillus sp2 fungi were found in rice plant tissues.Fungal identification refers to the book Illustrated Genera of Imperfect Identification of Fungi [15].The results of fungal reisolation are shown in Figure 4.The results of analysis of variance showed that the treatments were not significantly different from the control treatment.Figure 1 shows that the application of Aspergillus sp1.and Aspergillus sp2. was less able to inhibit P.oryzae isolates from the Mandai region.
Figure 2, the results of observations 7, 9 and 11 days after inoculation showed that the treatment of Aspergillus sp1 and Aspergillus sp2 fungi was also less able to inhibit blast disease attacks originating from the fungus P. oryzae from the Moncongloe area.While at 13 days after inoculation showed significantly different results.The treatment of Aspergillus sp1.and Aspergillus sp2.fungi on rice plants was able to inhibit the attack of blast disease caused by P.oryzae isolates from the Moncongloe region.Based on the table of leaf blast disease severity scale, with data at 13 days observation, it can be determined that the control treatment shows the criteria of highly susceptible and the treatment of P. oryzae with Aspergillus sp2.and treatment of P. oryzae with Aspergillus sp1.shows the criteria of susceptible.
Figure 3 shows that the treatment of Aspergillus sp1 and Aspergillus sp2 on observations on 7 and 9 day after inoculation were not able to inhibit blast disease attack as indicated by the results of variance analysis which had no significant effect, but on 11 and 13 days the results of variance analysis were significantly different.The treatment of Aspergillus sp1 and Aspergillus sp2 was able to inhibit P. oryzae from the Simbang region.Based on the table of leaf blast disease severity scale.[16] With data at 13 days observation, it can be determined that the control treatment shows the criteria of highly susceptible and the treatment of P. oryzae with Aspergillus sp2.and treatment of P. oryzae with Aspergillus sp1.shows the criteria of susceptible.The percentage inhibition of Aspergillus sp1 and Aspergillus sp2 fungi against blast disease caused by P. oryzae from different regions indicates that the isolates of P.oryzae species have different pathogenicity and virulence.The composition and dominance of P. oryzae races in a region can change depending on the growing season and ecosystem changes.Changes in P. oryzae races can be influenced by differences in rice cultivars grown in a region.[17].
Some researches mention that the fungus genus Aspergillus can function as a biological control against plant diseases.The application of Aspergillus fumigatus and Aspergillus terreus fungi to plants was able to inhibit blast disease attack.The results showed that the fungi A. fumigatus and A. terreus were able to inhibit the growth of P. oryzae and at 16 days after inoculation, the inhibition of A. fumigatus and A. terreus against P. oryzae reached more than 90%. A. terreus is an endophytic soil fungus that can enhance plant growth and show strong biocontrol activity [10].The fungus A. terreus can inhibit the growth of P. oryzae by competing for space and nutrients and is parasitic.A. terreus as a plant growth promoter and biocontrol microorganism.The fungus A. terreus isolated from soil able to suppress the pathogen Sclerotinia sp, is hyperparasitic and causes 100% mortality of S. sclerotium conidia.[11].While the fungus A. fumigatus can inhibit the growth of P. oryzae by means of competition, cell wall lysis, antibiosis and parasitism in suppressing the growth of P. oryzae.Fungi A. flavus, A. fumigatus, A. niger inhibit by competing, lysing the pathogen cell wall, and producing antibiotic substances called antibiotic mechanisms, in general the fungi A. fumigatus and A. terreus can be used as antagonists.Aspergillus sap. was a soil fungus that is very easy to find in the air.Some studies mention that genus Aspergillus is also able to live in plant tissues (endophytes).[18]

Conclusion
Aspergillus sp1 and Aspergillus sp2 fungi obtained from the rhizosphere of rice plants can suppress blast disease (P.oryzae) in vivo and have potential as biological control agents.

Fig. 2 .
Fig. 2. Percentage of blast disease intensity using P. oryzae isolates from Moncongloe region.Description Po2 : Rice plant with P. oryzae from Moncongloe region (control), Po2Asp1 : Rice plant with P. oryzae from Moncongloe region and Aspergillus sp1, Po2Asp2 : Rice plant with P. oryzae from Moncongloe region and Aspergillus sp2.Figure2, at 7 days observation showed the highest intensity is seen in the treatment of P. oryzae with Aspergillus sp2. was 4.67%, then followed by the treatment of P. oryzae with Aspergillus sp1. was 3.99% and the lowest intensity is seen in the control treatment was 3.76%.At 9 days , there was an increase in disease intensity in each treatment.The highest intensity was still seen in the treatment of P. oryzae with Aspergillus sp2. was 6.39%, then in the control treatment was 5.70% and the lowest intensity was shown in the treatment of P. oryzae with Aspergillus sp1. was 5.52%.However, at 11 days,the control treatment exhibited the highest intensity (7.67%), followed by the P. oryzae treatment with Aspergillus sp2.(6.63%) and the P. oryzae treatment with Aspergilus sp1 (5.98%).Observations at 13 days showed that the highest intensity was seen in the control treatment ( 9.00%) , then in the treatment of P. oryzae with Aspergillus sp2 was 7.00% and the lowest intensity in the treatment of P. oryzae with Aspergillus sp1. was 6.00%.

Fig. 3 .
Fig. 3. Percentage of blast disease intensity using P. oryzae isolates from Simbang region.Description Po3 : Rice plant with P. oryzae from Simbang region (control), Po3Asp1 : Rice plant with P. oryzae from Simbang region and Aspergillus sp1, Po3Asp2 : Rice plant with P. oryzae from Simbang region and Aspergillus sp2.Figure3, 7 days observation showed the highest intensity is shown in the control treatment was 5.88%, then followed by the treatment of P. oryzae with Aspergillus sp1. was 4,21% and the lowest intensity of P.oryzae with Aspergillus sp2 was 4,02%.At 9 days , there was an increase in disease intensity in each treatment.The highest intensity is shown in the control treatment at 7.35% then in the treatment of P. oryzae with Aspergillus sp1 at 6.05%, and the lowest intensity was seen in the treatment of P. oryzae with Aspergillus sp2. at 5.83%.At 11 days, the highest intensity of blast attack was in the control treatment at 8,00%, then in the treatment of P. oryzae with Aspergillus sp1 at 6.00%, and also the treatment of P. oryzae with Aspergillus sp2 at 6.00%.Observations at 13 days showed the highest intensity in the control treatment was 8.00%, then the treatment of P. oryzae with Aspergillus sp2. was 6.00% and the treatment of P. oryzae with Aspergillus sp1 was 6.00%.