Effect of Bacterial Consortium and Rice Husk Charcoal to Control Downy Mildew in Corn (Zea mays L.)

One of the obstacles in cultivating corn is downy mildew caused by the fungus Peronosclerospora. Downy mildew is one of the primary diseases of maize caused by Peronosclerospora philliphinensis. Plants infected with downy mildew will not give optimal results. Typical symptoms of downy mildew disease in corn plants are chlorosis extending parallel to the veins of the leaves, stunted growth of the affected plantsand a layer of white powder under the surface of the leaves in the morning. Downy mildew pathogens encompass various species of obligate oomycetes, posing severe threats to cultivated, ornamental and indigenous plant populations (Salcedo et al., 2021). Species such as Peronosclerospora sorghi, P. philippinensis, P. sacchari, P. spontanea, Sclerophthora rayssiae var. zeae and P. maydis have caused substantial losses due to widespread downy mildew outbreaks worldwide (Janruang and Unartngam, 2018). Corn plants infested with P. maydis can experience a reduction in production of 80% -100%. Corn plants attacked by P. maydis cannot produce seeds (Herlina et al., 2017).
       
Control of downy mildew disease generally uses seed treatment with synthetic fungicides. However, the excessive use of synthetic fungicides can result in the emergence of novel strains and outbreaks within agricultural environments, thereby adding complexity to disease control efforts. Consequently, relying on synthetic fungicides over prolonged periods is not environmentally sustainable and may prove ineffective as a long-term strategy for mitigating downy mildew disease (Wu et al., 2023). The continued use of synthetic fungicides is known to have a negative impact on non-target organisms and the soil. Using biological agents is a solution to eliminate fungicides in combination with resistant varieties (Najamuddin et al., 2023). Other control techniques with resistant varieties continue to be developed. However, no varieties have been reported to be highly resistant to downy mildew attack (Arulselvi and Selvi, 2021). There is a need to develop other control methods, such as bacteria. 
       
Using rhizobacteria is an alternative to control downy mildew in corn plants caused by Peronosclerospora maydis. Rhizobacteria are a group of bacteria with a habitat in the plant root area (rhizosphere) which has been widely researched and proven to increase soil fertility, increase plant resistance and suppress plant pathogens (Wang et al., 2021). Rhizobacteria act directly as biofertilizers and bio-stimulants by producing plant growth hormones such as IAA (Indole -3- acetic acid), gibberellin, cytokinin,  ethylene, dissolving mineralsand indirectly also function to prevent pathogenic microorganisms through the formation of siderophores, antimicrobial components (antibiotics) and potentially as an agent in induced systemic resistance (Purnawati et al., 2023). Applying biological agents to control downy mildew is an environmentally friendly method recently widely promoted. Trichoderma sp. and plant growth promoting rhizobacteria (PGPR). The results showed that the combination of Trichoderma sp. and rhizobacteria of 60 mL L-1 was able to inhibit the incidence of the disease up to 66.53% and the disease severity up to 89.84% (Purwant, Wahyudiand Hamyana, 2023).
       
Utilization of bacterial isolates obtained from the Rhizosphere of dry land roots of annual plants (Coconut Rhizosphere (Cocos nucifera), Pine Rhizosphere (Casuarina equisetifolia), Cassava Rhizosphere (Manihot esculenta), Shrub Rhizosphere (Chamaedaphane), Sono Rhizosphere (Dalbergia latifolia), Banana Rhizosphere (Musa paradisiaca) (Sukorini et al., 2023). The results of initial isolation and selection to determine the types of soil bacteria that play a role and have the potential to be developed and explicitly utilized obtained the ten best bacterial isolates from the Rhizosphere of annual plants. The ten bacterial isolates obtained were subjected to a hypersensitive test using tobacco plantsand seven antagonistic bacteria were obtained. This bacterial isolate was then formulated by mixing the seven bacteria with antagonists to form a bacterial consortium. This is because there is a mechanism between bacteria that support each other to inhibit pathogens that attack plants. This is based on the statement (Jahuddin et al., 2022) that bacterial consortia have better inhibitory power than pure isolates of individual antagonist bacteria in inhibiting the development of pathogens. The application of a bacterial consortium will colonize plants in different ways depending on how it is applied; according to (Limbo-dizon et al., 2023), the application of a bacterial consortium must be as close as possible to the plant because the inoculated bacteria can only move a distance of no more than 2 cm from the inoculation location. Incorporating silica derived from rice husk charcoal has been discovered to have a notable impact in preventing harmful fungal infections, specifically downy mildew, which affect corn plants (Senapati et al., 2022). The treatment of giving organic silica derived from rice husk ash was able to suppress downy mildew attacks on corn plants caused by P. maydis, with levels only reaching a value of 4.63 and a disease incidence of 11.11%.

This research was conducted during the rainy season from February to June 2023. It is located in the Agrotechnology Farm of the University of Muhammadiyah Malang at an altitude of 560 meters above sea level, with the coordinates 7°55’04" S 112°35’41" E.

Experimental Setup
 
This study used a randomized block design (RBD) arranged factorially. There are two factors: The first factor was Charcoal husk. The second factor was bacterial consortium application. The treatment details were: Charcoal husk (H): H5 = Charcoal husk 5 tons/ha, H10= Charcoal husk 10 tons, H15 Charcoal husk 15 tons/ha. Bacterial consortium (W): W0 = Without bacteria, WL = Bacterial consortium 10cc/l via foliar spraying, WS =Bacterial consortium 10 cc/l through the soil, WLS = Bacterial consortium 10 cc/l through the soil and foliar spraying
 
Bacterial consortium preparation
 
Each bacterium was cultured in a Potato Dextrose Agar (PDA) medium for five days. Then transfer to Potato Dextrose Broth (PDB) medium. The bacteria population was counted using optical density (OD) measuring. Each bacterial isolate was taken in as much as 1 mL and distilled water was put into a cuvette and used as a blank. Suspension The absorbance value was then calculated with a spectrophotometer wavelength of 649 nm (Hyder et al., 2024). Observations were made at four-hour intervals from the start of the observation and carried out for 24 hours.
 
Land preparation and cultivation practice
 
The land was cleared of weeds and crop residues with a hoe to loosen the soil. Then, the loose soil was made into elongated beds in an east-west direction. Beds were made with a height of 20 cm, 2.2 m long and 1 m wide and irrigation channels were made 30 cm wide.  Sweet corn seeds of the Bonanza F1 variety were planted in the planting holes made using a drill in each plot with a spacing of 70 cm ´ 40 cm and a depth of approximately 5 cm in each plant hole with two seeds of corn. Fertilization with charcoal husk was carried out at the beginning of planting corn seeds at a dose of five tons/ha (140 g/plant). Ten tons/ha (240 g/plant). Fifteen tons/ha (420 g/plant). Charcoal husk was applied by sprinkling rice husk charcoal in the planting hole.
 
Provision of the bacterial consortium
 
Bacterial consortium application was carried out at plant ages 10, 17, 24 and 31 days after application (DAA) with a dose of 10 ml/plant and a bacterial density concentration of 108 cfu/ml (Hadi et al., 2021). The bacteria was sprayed on all parts of the corn plants that were still healthy.
 
Observational variables
 
Observational variables consist of number of leaves, plant length, stem diameter, cob length, cob diameterand disease intensity (DI). DI was done by observing the infection or damage that occurs in plants. Calculation of the disease intensity using the following formula:
   
Where:
I=  Intensity of severity of the disease.
a=  Number of plants affected by downy mildew.
b=  The total number of plants.
 
Statistical analysis
 
Statistical analyses of data were carried out for Randomized Block Design. Data were subjected to the analysis of variance and least significant difference at a probability level d” 0.05. The non-significant differences were denoted as ns.

Number of Leaves
 
There was no interaction effect between the husk charcoal dose and the bacterial consortium application. The application of husk charcoal and the bacterial consortium had no significant impact on the number of leaves observed 7, 14, 21, 28 and 35 days after application (DAA, presented in Table 1).
 

 
Plant height
 
There was no interaction effect between the husk charcoal dose and the bacterial consortium application. Providing husk charcoal had no significant effect on plant height. Likewise, the application of bacterial consortium except at the age of 7 days after application (DAA). They are presented in Table 2.
 

 
Stem diameter
 
There was no interaction effect between the husk charcoal dose and the bacterial consortium application. As presented in Table 3, the provision of husk charcoal and the bacterial consortium had no significant impact on the stem diameter of corn plants.
 

 
Cob diameter, cob length and disease incidence
 
Charcoal husk treatment without bacterial consortium did not affect cob diameter and length. This is in line with the percentage of disease incidence (%). disease incidence occurred in the rice husk charcoal treatment alone without the bacterial consortium. The intensity of the attack reaches more than 90% at the age of the plant 70 days after application. The lowest intensity of attack was in the combination treatment of 15 tons of husk charcoal per hectare and bacterial consortium by foliar and soil spraying. In treatments that were attacked earlier, namely, in treatments without the addition of bacterial consortia, no cobs were produced. This is presented in Table 4 and Fig 1.
 

 

       
The performance of corn plants given rice husk charcoal and bacterial consortium showed a lower attack level than those given rice husk charcoal alone. This has an impact on crop yields. Giving husk charcoal can bind nutrients well to encourage plant growth. Adding husk charcoal to soil media can improve the porosity of soil media, so it is suitable for root respiration and soil moisture (Asfar et al., 2021). According to (Syamsafitri et al., 2023) nutrient content on rice husk charcoal includes several components namely SiO₂ (52 Percent), C (31 Percent), K (0.3 Percent), N (0.18 Percent), F (0.08 Percent) and calcium (0.14 Percent, besides that there are also other components such as Fe₂O₃, K₂O, MgO, CaO, Mn and Cu in small quantities. Besides that, rice husk charcoal can maintain environmental conditions and improve the physical properties of the soil, making the water-holding capacity high so that vegetative growth is a better corn crop. The application of rice husk charcoal to the soil, besides being able to be light and high porosity, the soil remains loose, retains soil moistureand stores components nutrients, driving the growth of organisms that are useful for plants and as component anchors nutrient crops when plants are deficient nutrientand husk charcoal is referred to as fertilizer slow release (Bayu Murti Petrus et al., 2020). The ability of the silica component contained in the husk charcoal. The presence of Sufficient silica in the soil can help plants to be more resistant to component imbalances nutrients, which include excess N, deficiency and abundance of Pand poisoning of Na, Fe, Mn and Al (Katz et al., 2021).
       
The resistance level of maize plants treated by the bacterial consortium also indicated that the intensity of the attack by the pathogen Peronosclerospora was low. This is because the bacterial consortium, when formulated, has a disease control mechanism; the treatment of this antagonistic bacterial consortium provides a defense system (bioprotectant) because these bacteria can secrete antibiosis compounds which can encourage attacked plants to fight pathogens (Mugiastuti et al., 2023). This bacterium is thought to be able to produce compounds, such as antibiotic compounds, siderophoresand other secondary metabolites whose properties can slow down fungal activity. Inhibition of pathogens, namely bacteria, can fight against the invasion of phytopathogens by producing siderophores. Siderophore is an iron chelating compound or Fe3+ complex made by microorganisms to hide the iron micronutrient components around the rhizosphere so that pathogenic microorganisms cannot access it (Pecoraro et al., 2022).
       
It is suspected that this consortium contains several microorganisms that are good for agriculture to increase the productivity and growth of corn plants. Like (Miljakoviæ et al., 2024), plant nutrients may also contribute to the higher efficacy of microbial inoculants as well as the growth and development of plants. Moreover, these organisms exhibit the capability to colonize plant roots, providing numerous benefits to their hosts, including the modulation of phytohormone production, enhancement of soil nutrient availabilityand bolstering resistance against pathogens.
       
Furthermore, their presence minimizes the reliance on chemical fertilizers, mitigates both biotic and abiotic stress factorsand ultimately boosts crop production, as highlighted (Asghari et al., 2020). Among the microorganisms utilized to augment agricultural productivity are Azospirillum, Arthrobacter, Bacillus, Burkholderia, Enterobacter, Flavobacterium, Pseudomonas, Rhizobium, Frankia, Klebsiella, Clostridium, Trichoderma, Beauveria, Serratia and Streptomyces, as outlined (Jeane and Dias-filho, 2021).
       
Nitrogen is a vital macronutrient essential for protein and nucleic acid synthesis. Microorganisms like Azospirillum, Azotobacter, Achromobacter, Bradyrhizobium, Beijerinckia, Rhizobium, Clostridium, Klebsiella, Anabaena, Nostocand Frankia play a significant role as biological nitrogen fixers, converting atmospheric nitrogen gas (N₂) into ammonia (NH₃) (Bhat et al., 2019). This bacterial consortium possesses the ability to assimilate nitrogen-fixing microorganisms, facilitating nutrient recycling, particularly for phosphorwiSus (P) and potassium (K), while also stimulating plant growth. The presence of nitrogen (N) and phosphorus (P) components fulfils the nutritional requirements of plants. The P component is essential for plants to affect photosynthesis and determine harvest age.
 
Throughout the process of seed filling, seeds assimilate nutrients from the leaves and other plant tissues. These acquired nutrients serve as essential components for a multitude of metabolic pathways, encompassing protein synthesis, starch formationand other biochemical processes vital for seed development. Concurrently, seeds undergo notable metabolic transformations, including the activation of enzymes engaged in starch metabolism, facilitating the storage of energy in the form of starch. As seeds reach maturity, there is an increased accumulation of metabolic by products, contributing to the attainment of maximum size and weight in the produced seeds (Shaw and Cheung, 2021).
       
The number of leaves and plant height were not affected by those treatments. This is presumably because internal factors influence the number of leaves. In line with research (Herhandini et al., 2021), the provision of rice husk charcoal and the consortium bacteria does not affect the number of leaves on corn plants because plant leaves are more due to plant genetics. According to (Jeane and Dias-filho, 2021), components affecting plant growth, the number of leaves is genetically determined by abiotic factors such as soil (nutrients, heavy metals, pH and salinity), water availability, light intensityand temperature can affect plant-microbe interactions, as they can alter plant metabolism, root exudate composition and rhizosphere biology. This is because, if changes in plant exudative patterns occur, the same treatment and the same plant genotype may interact differently.

The intensity of downy mildew attack on the combined application of the bacterial consortium and rice husk charcoal was lower compared to no combination treatment. The length and diameter of plants treated with the rice husk charcoal plants had a significant effect. Still, the application of the bacterial consortium had no effectand the number of leaves was not affected by the treatment.  To control downy mildew disease, 15 tons/ha of husk charcoal can be used in combination with consortium bacteria applied to leaves and soil.

All authors declare that they have no conflicts of interest.