Controlling of downy mildew on maize using a combination of varieties and mycorrhizae

Downy mildew is one of the main diseases of maize caused by Peronosclerospora philliphinensis. Plants infected with downy mildew will not give optimal results. Downy mildew is commonly controlled by a combination of resistant varieties and systemic fungicides. The continued use of systemic fungicides is known to have a negative impact on non-target organisms and the soil. The use of biological agents is a solution to eliminate the use of fungicides in combination with resistant varieties. This study aimed to evaluate the effectiveness of combining varieties and arbuscular mycorrhizal fungi (AMF) in controlling downy mildew on maize. The study was conducted in greenhouse conditions, using a randomized block design with twelve combinations of varieties, mycorrhizal and inoculation of pathogen treatments, namely V1M0P0, V1M0P1, V1M1P0, V1M1P1, V2M0P0, V2M0P1, V2M1P0, V2M1P1, V3M0P0, V3M0P1, V3M1P0, and V3M1P1. V1 Annoman variety (susceptible check), V2 JH29 variety (moderate) and V3 Pertiwi 6 variety (resistant check). Each treatment was repeated five times, with two plants in each experimental unit. All treatment combinations carried out observations of plant height, number of leaves, and diameter. Observations of disease incubation, incidence, and severity of disease were carried out on six treatment combinations inoculated with the P. philliphinensis. The results showed an increase in plant vegetative growth in the combination treatment of varieties and mycorrhizae in the absence of pathogen infection (V1M1P0, V2M1P0, and V3M1P0). The disease incidence and severity suppression of downy mildew was shown in the combination V3M1P1 treatment with the same incidence of 50% and disease severity of 46% at 28 days post inoculation. The results of this study indicate the need for the right combination of controls for the purposes of effective disease management in plant cultivation.


Introduction
Downy mildew is the main disease of corn plants.Plants that are attacked by downy mildew will not give optimal results.A study revealed that downy mildew in maize could reduce yields by up to 90% [1].Infection at the beginning of growth in susceptible varieties causes greater yield losses.In Indonesia, downy mildew is caused by three Peronosclerospora species, namely P. maydis, P. sorghi, and P. philippinensis.P. philipinensis is the dominant species in North Sulawesi and South Sulawesi [2][3][4].1230 (2023) 012100 IOP Publishing doi:10.1088/1755-1315/1230/1/012100 2 Downy mildew control in maize is mostly done by combining resistant varieties with fungicides that are applied at the beginning of planting.The use of resistant varieties and metalaxyl fungicides applied through seed treatment is effective in controlling downy mildew [1].The use of fungicides in the long term can have a negative impact.Harmful to non-target organisms, causing structural compaction, residues that are difficult to decompose and present with risks to consumer health [5].These risks lead to the need for alternative controls that are environmentally friendly and do not pose a health risk to consumers.The use of resistant varieties on a large scale without sustainable cultivation is extremely risky, resulting in the formation of new strains.The new strains will undermine the plant's resistance.To anticipate, it is necessary to combine environmentally friendly control techniques with low health risks.Resistance properties can be enhanced through elicitor-induced induction by involving the coordination and expression of certain genes characterized by the accumulation of certain compounds such as salicylic acid, jasmonic acid and ethylene as well as the expression of proteins related to pathogen pathogenesis [6,7].Induction by elicitor can also be carried out using microorganisms with special characteristics, such as endophytic microbes, philosphere microbes, nonpathogenic microbes, antagonistic microbes, and plant growth-promoting microbes such as PGPR, PGPF, and mycorrhizal fungi [8][9][10].
Arbuscular mycorrhizal fungi (AMF) are very abundant and have considerable potential to suppress plant diseases.AMF is abundant in soil and helps plants in growth, development, stress tolerance, soil pollutant remediation, C-sequestration, crop resistance, and agricultural sustainability.AMF helps plants in the absorption of nutrients by expanding the hyphal network around the rhizosphere.Mycorrhizal infection changes the root architecture and causes the absorption of nutrients from the roots to be much better [11].
Research on the ability of AMF to increase plant resistance has been reported in several studies.A study [12] reported that AMF was able to induce resistance of wheat plants to Blumeria graminis disease.B. graminis infection on wheat leaves was reduced by 78% in plants with the mycorrhizal application.AMF is effective in increasing plant tolerance to biotic stress.The effectiveness depends on the conditions of biotic and abiotic factors.The underlying mechanisms include competition for host nutrients, space, and photosynthesis, changes in the rhizosphere, and induction of host defenses [13].AMF is effective in plant protection against Verticillium spp.The effectiveness of AMF in protecting its host plants varies greatly depending on environmental factors such as the concentration of carbon dioxide in the atmosphere, availability of groundwater, and air and soil temperatures.Increased resistance by mycorrhizae is caused by improved nutrient and water status of plants as well as increased antioxidant metabolism and/or increased production of secondary metabolites in plant tissues [14].
This study aims to determine the effectiveness of the combination of resistant varieties and AMF in controlling downy mildew on maize.Until now, information regarding the effectiveness of the combination of resistant varieties and AMF against downy mildew caused by Peronosclerospora spp.has not been widely carried out.The absence of a report on the results of this study makes this research important because it can be an alternative solution for controlling downy mildew on corn that is environmentally friendly and practical in its application.

Preparation of mycorrhizal isolates
The Arbuscular Mycorrhizal Fungi (AMF) used is a product of the Southeast Asian Regional Center For Tropical Biology (SEAMEO BIOTROP) Glomus etunicatum.Before use, the spores were observed (re-checked) on the zeolite carrier media.

Preparation of planting materials and inoculum propagation
Planting material used consisted of 60 units of 30x40 cm polybags, planting media in the form of soil mixed with manure in a ratio of 2:1 (v/v) sterile soil through soil cooking, corn seed, zeolite media and mycorrhizal inoculum source.Annoman variety (vulnerable) as a propagation plant, then inoculated with a source of inoculum obtained from the location around the study to be inoculated on multiplication plants.The inoculum obtained was collected on susceptible maize plants that had been planted before the study was carried out and prepared for the preparation of inoculation to plants during the testing phase.Maintenance of plant sources of inoculum is done by watering, weeding and fertilizing.Watering is done by paying attention to soil moisture conditions and plant needs.Fertilization is done using Phonska NPK fertilizer at a dose of 300 kg/ha and Urea fertilizer at 200 kg/ha.The first fertilization is given when the plants are ten days after planting with a volume of 300 kg/ha phonska and 100 kg/ha Urea.The second fertilizer was given when the plant was 28 days after planting with a volume of 100 kg/ha urea.Weed control is carried out physically by removing weeds that grow in polybags and around the test plants.

Maize planting and mycorrhizal inoculation
Corn seeds of Annoman (Cek Vulnerable), JH-29, and Pertiwi-6 varieties were washed and rinsed using sterile water and then dried.Furthermore, the seeds were planted in polybags measuring 30x40 cm, which already contained soil and manure (2:1).Each polybag was filled with two seeds and planted together with the mycorrhizal application using 20 grams of zeolite carrier media.Plants were given a barrier in the form of mica plastic that was circular on each test plant.Plants are maintained and watered every morning and evening.The corn plants used were corn plants with uniform criteria and free of disease before inoculation.

Inoculation of pathogens
Inoculation of pathogens in corn plants was carried out when the plants were 10 days after planting.Inoculation was carried out at 03.30 am.Inoculation was carried out by the method of suspension spraying and symptomatic leaf attachment [15].The source of inoculum was obtained from the inoculum collection that had been propagated previously in a separate place with the test plant.

Re-Identification of mycorrhizal species
Identification of mycorrhizal species was carried out by observing the mycorrhizal structure.The variables observed were the morphological characters of the spores, which included the shape, color, and layer of the spores [16].

Plant agronomic observations
Observations of plant height, number of leaves and stem diameter were carried out at 14 and 28 days post pathogen inoculation.Plant height was measured from the base of the stem to the highest part of the stem.The number of leaves was measured from the lowest leaf to the fully opened upper leaf.Stem diameter was measured at the lowest internode of the three plants with the widest circumference of the stem.

Observation of incubation period and disease intensity
Observation of disease development was carried out by observing the incubation period, Incidence and Severity of downy mildew.Observations of the incubation period (the time the symptoms appear post inoculation to the host) and the Incidence of disease are carried out every day starting from the time the pathogen is inoculated into the corn plant until the plant causes disease symptoms.Severity observations were carried out twice, namely 14 and 28 days post pathogen inoculation The Incidence of the disease is calculated by the formula: The severity of the disease is calculated by the formula:

Data analysis
The research data were analyzed descriptively and quantitatively, and analysis of variance using Ms.
Excel and Minitab V.20.The analysis of variance results were significantly different, followed by Fisher's Post hoc test at the 95% confidence level.

Re-identification of mycorrhizal spore types
Arbuscular mycorrhizae fungi (AMF) are natural organisms that contribute to productivity and ecosystems and are effective in plant disease management.Re-identification of AMF Claroideoglomus etunicatum was carried out by looking at the appropriate color and size [17] based on information from related research results (Figure 1).Claroideoglomus etunicatum is characterized by round, oval, to ellipsoid-shaped spores with a diameter of 40-135 µm, pale yellow, dark yellow to brown, sometimes ovoid with a smooth to granular surface and two-layered spore walls [18][19][20].

Plant agronomic observations
Plant phenotype is the result of the interaction between the genotype and the environment.Genetic potential and suitable conditions contribute greatly to the expression of agronomic characteristics of plants, including height, the number of leaves, stem diameter and response of plant resistance to diseases caused by biotic and abiotic factors.Biotic disease disorders are caused by infectious agents that directly play a role in the agronomic character of the host plant.The results revealed that the combination of varieties and AMF affected the agronomic expression of plants.The effect is in the form of differences in plant height, the number of leaves, stem diameter, and plant response to pathogens.In general, the results of observations for each variety, AMF infection, and corn downy mildew infection, gave different results for each agronomic character.Observation of plant agronomic characters showed the best results in the combination of varietal and AMF treatments without pathogen infection (Table 1).The best results were obtained on V2M1P0, V3M1P0, and V1M1P0.V2M1P0 has the best value from all observed agronomic aspects, such as plant height, number of leaves, and stem diameter.V3M1P0 had plant height and stem diameter values that were not significantly different from V2M1P0 treatment, while V1M1P0 had leaf number values that were not significantly different from V2M1P0.The best plant height results for observations of 14 and 28 day post inoculation (dpi) were obtained in the V2M1P0 treatment with an average value of 41.12 cm and 111.86 cm.This value is significantly different from the observed values obtained in the V1M0P1, V1M0P0 and V2M0P1 treatments with an average value of 76.20 cm, 86.30 cm, and 91.20 cm (table 1).Number of Leaves, there was no significant difference between treatments on the observation of the number of leaves 14 dpi.Different results were obtained by observing the number of leaves 28 dpi.The highest number of leaves was obtained in the V2M1P0 treatment, with an average number of 9.9 leaves.This value was not significantly different from the V2M0P0 treatment which had an average number of leaves of 9.8.The number of leaves was significantly different from the V1M0P1 treatment with an average leaf number of 8.83 leaves (table 1).The best 14 dpi stem diameter was obtained in the V2M0P0 treatment with a value of 18.50 mm.This result was not significantly different from the value of the stem diameter in the V3M1P0 treatment of 18.40 mm.Slightly different results on observations 28 dpi.The best treatment was obtained at V3M1P0 with an average diameter of 19.17 mm, not significantly different from the V3M0P0 treatment with an average diameter of 19.10 mm.AMF is a biotrophic agent that provides a supply of nutrients and water to the host with a symbiosis of the utilization of photosynthetic products by AMF.Therefore, AMF is reported to increase nutrient uptake and protect plants from pathogens [21].Several studies report AMF as an effective biofertilizer for sustainable agriculture.AMF colonization increases phosphorus uptake and plant tolerance under stress conditions by eliciting several changes in plant morphology and physiology [22][23][24].AMF acts as a natural growth regulator for plants due to its growth-stimulating capacity [25].AMF symbiosis can significantly increase root strength, promote nutrient accumulation, and affect nutrient distribution, and these changes are significantly correlated with changes in root exudate [26].Mycorrhizal inoculation significantly increased plant height and stem circumference, and the use of commercial AMF inoculants has the potential to promote soybean growth and yield under suitable environmental conditions [27].Glomus species, individually and in combination with other treatments, can increase the agronomic growth of plants [13].Mycorrhizal inoculation significantly increased plant height, stem circumference, leaf number, and leaf area ratio of soybeans compared to plants without AMF.The increase in growth can reach 26% [28].Increased vegetative growth indicates better yield potential under suitable conditions [27,29].The increase in plant growth is thought to be due to the ability of AMF to have a stimulating effect on plant growth, increase the range of nutrient uptake, and increase nutrient P and air transport [23,30,31].1230 (2023) 012100 IOP Publishing doi:10.1088/1755-1315/1230/1/0121006 P. philliphinensis pathogen infection has a significant effect on the agronomic growth of plants.In the presence or absence of AMF infection, pathogen infection remains the main factor in the difference in the value of plant morphology observations.For example, the V2M1P0 and V2M0P0 treatments had better agronomic values for plant height, leaf number, and stem diameter compared to V2M1P1 and V2M0P1 treatments.The same thing was also obtained in the observation of 28 dpi (table 1).Pathogens interfere with the process of photosynthesis and interfere with the flow of nutrients from leaf cells into the phloem, while in the phloem, pathogens develop into other plant tissue cells, resulting in stunted plants.P. philippinensis infection is systemic, infects all plant tissues and in susceptible varieties, causes stunted growth.In susceptible varieties (Anoman), P. philippinensis infection occurs earlier so that the plants are stunted compared to healthy plants [1].

Incubation period, incidence, and severity of disease
The incubation period of a disease is the time lapse between exposure to the infectious agent causing the disease and the onset of symptoms (clinical) of the disease [32].Varieties have a significant effect on suppressing pathogen infection in plants.The results showed that the pathogen P. philliphinensis only took five days post inoculation (dpi) to infect and cause symptoms in maize varieties of annoman (susceptible check) under screen house conditions.In contrast to other varieties, the pathogen took at least more than 7 days to infect the JH-29 variety, and even more than 12 days to infect the pertiwi-6 variety (check for resistance) (Figure 2).The difference in the incubation period of the disease in several treatments was caused by differences in varieties and the presence of mycorrhizae.The longer the incubation period of the disease against a condition indicates the presence of factors inhibiting the early development of the pathogen.In plant management using biological agents, the induction of resistance and varietal resistance properties can be the main obstacle to the development of pathogens to cause disease [7,10,12].Each variety has a different level of resistance to pathogens [1,8].Induction of resistance arises due to the interaction of pathogen, host, and environment [7,25].Information regarding the incubation period of the pathogen can be used as the basis for epidemic modeling for the evaluation of overall disease control strategies [32].The lowest incidence and severity of downy mildew at 14 dpi and 28 dpi were observed in the combination treatment V3M1P1.The results of the observation of the Incidence of V3M1P1 treatment were 10% and 50%, respectively, with disease severity of 2% and 22% at the observations of 14 dpi and 28 dpi.These results indicate a significant suppression of disease incidence when compared to the V1M0P1 treatment (susceptible control without mycorrhizae).Suppression of disease incidence by 90% and disease severity by 34% at 14 dpi observation and suppression of disease incidence by 50% and disease severity by 46% at 28 dpi observation (figure 2).
The variety factor has a very significant effect on suppressing the Incidence and Severity of downy mildew on maize.The Incidence and Severity of downy mildew in the V3M0P1 treatment observed at 14 and 28 dpi had a very low value when compared to the V1M0P1 treatment.V3M0P1 had disease incidence values of 10% and 60% with disease severity of 8% and 22% at 14 dpi and 28 dpi observations, respectively.The results of the V3M0P1 treatment showed that there was a suppression of disease incidence by 70% and disease severity by 28% at 14 dpi observations and suppression of disease incidence by 40% and disease severity by 46% at 28 dpi observations (figure 3).In addition to the variety factor, the AMF factor also contributed to the Incidence and Severity of downy mildew in the three maize varieties tested.The Incidence and Severity of downy mildew in the treatment of V1M1P1, V2M1P1, and V3M1P1 at observations 14 and 28 dpi had lower values when compared to the Incidence and Severity of disease in the V1M0P1, V2M0P1, and V3M0P1 treatments.V1M1P1 had disease incidence values of 60% and 100% with disease severity of 26% and 62% at 14 dpi and 28 dpi observations, respectively.This result is better than the V1M0P1 treatment with a disease incidence of 80% and 100% with disease severity of 36% and 68% at 14 dpi and 28 dpi, respectively.The results of the above comparison show suppression of disease incidence by 20% at 14 dpi and disease severity by 10% and 6% at 14 dpi and 28 dpi observations.The complete results regarding the effect of the combination of varieties and AMF, the effect of varieties and AMF singly on the suppression of the Incidence and Severity of downy mildew in the three tested maize varieties are presented in Figure 3. Downy mildew-infected plants have stiff leaves and appear stunted compared to plants not infected with downy mildew.On the underside of infected leaves are piles of conidia-like whitish powder (figure 4a and 4b).Visually, downy mildew caused by Peronosclerospora species is difficult to distinguish, so molecular detection technology is recommended to improve the efficiency and effectiveness of disease detection [1][2][3].Resistant varieties mostly control downy mildew.Each variety has inherited natural resistance, can be induced by biological agents, and has real effectiveness in controlling plant diseases [6,10,33].The application of endomycorrhizal fungi is proven to increase plant resistance to several root diseases due to the effects of mycorrhizal fungi on vegetative growth processes such as absorption of soil nutrients, dissolving phosphate through the formation of phosphatase enzymes and increasing photosynthesis through protecting plant health [11,26,30].Mycorrhizal fungi also change root morphology and expand the range of uptake of nutrients and water by external mycelia [11].

Conclusion
The results showed that the combination of varieties and AMF effectively suppressing the incidence of downy mildew in maize.Varieties have a more significant effect on independent control compared to AMF.The effectiveness of AMF in increasing plant resistance is highly dependent on environmental conditions, including its interaction with host plants and target pathogens.For disease control, we suggest that the combination of varieties and AMF can be carried out using AMF of other species and isolates to obtain a better combination of varieties and AMF.The combination of varieties and AMF for plant disease control is also recommended for plant diseases with infection, not at the beginning of growth.Pathogens that are infected at the beginning of growth are still difficult to control using AMF, which takes time to infect plants before they can interact optimally.

I
= Incidence of plant diseases; a = number of symptomatic plants; b = number of plants observed.

S
= Severity of the disease, n = number of plants in the same attack symptom, v = scale value for each category of attack symptoms, Z = the highest scale value of the attack symptom category; and N = number of plants observed.The scale is given by observing the top 5 leaves on each test plant and scoring each leaf with the following criteria: 0: No infection/symptoms of disease, 1: Very mild infection/symptoms, about 1-5% on the leaves, 2: Mild infection/symptoms about 6-20% on the leaves, 3: Moderate infection/symptoms around 21-40% on leaves, 4: Severe infection/symptoms around 41-75% on leaves, 5: Very severe infection/symptoms greater than 75% of the leaves.

7 Figure 2 .
Figure 2. Diagram of the observation of the incubation period of the disease.

Figure 3 .
Figure 3. Diagram of the incidence and severity of downy mildew on observations of 14 dpi and 28 dpi on corn plants.

Figure 4 .
Figure 4. a. Stunted plants caused by Peronosclerospora sp., b. White flour on the leaf surface is a sign of downy mildew infection, c.Arbuscular Glomus etunicatum on infected plant root tissue.

Table 1 .
Observations on plant height, number of leaves, and stem diameter of 14 dpi and 28 dpi.Numbers in the same column followed by the same letter are not significantly different at Fisher's Post hoc test at the 95% confidence level