Potency of Non-Fungicide Chemicals for Maize Inducing Resistance against Downy Mildew

Downy mildew is one of the important diseases in maize cultivation in Indonesia. Wijaya (2010) stated that Peronosclerospora maydis, the causal agent of downy mildew, attacked 200 Ha maize planting areas in Tegal Regency, Central Java Province, Indonesia. Until now, seed treatment is still the one option to control as the serious infection occurred in seedling stage. Metalaxyl is an active ingredient commonly used for maize seed treatment in Indonesia. It is an A1 class pesticide due to the fungicide resistance action committee (FRAC), which is assumed to have a high risk in pathogen resistance (FRAC, 2017). Long term use of fungicides caused P. maydis resistance (Isakeit & Jaster, 2005; Sumardiyono, 2008). Based on this fact, an environmentally friendly method for controlling downy mildew is required. Plant induced resistance is a method to activate self resistance in plants by giving inducer materials which trigger the plant defense mechanism. Therefore plants become more resistant against subsequent pathogen infection (Conrath et al., 2002; Agrios, 2005; Faoro et al., 2008). External inducing materials (elicitors) can be biological, chemical and physical agents (Agrios, 2005; Widiastuti et al., 2013). According to Faoro et al. (2008), resistance induction offers several important advantages, such as it is not based on a modification of plant genomes, relatively cheap, it has no environmental risks; and effective against various plant pathogens. Research using non-fungicide chemicals as inducers of plant diseases in Indonesia has not been much conducted (Habibullah et al., 2018), especially on maize. In previous research, Habibullah et al. (2018) reported that maize seed dipping treatment by nonfungicide chemicals such as benzoic acid, sodium benzoic, salicylic acid or saccharin induced early response of plant defense by forming reactive oxygen ABSTRACT


INTRODUCTION
Downy mildew is one of the important diseases in maize cultivation in Indonesia. Wijaya (2010) stated that Peronosclerospora maydis, the causal agent of downy mildew, attacked 200 Ha maize planting areas in Tegal Regency, Central Java Province, Indonesia. Until now, seed treatment is still the one option to control as the serious infection occurred in seedling stage. Metalaxyl is an active ingredient commonly used for maize seed treatment in Indonesia. It is an A1 class pesticide due to the fungicide resistance action committee (FRAC), which is assumed to have a high risk in pathogen resistance (FRAC, 2017). Long term use of fungicides caused P. maydis resistance (Isakeit & Jaster, 2005;Sumardiyono, 2008). Based on this fact, an environmentally friendly method for controlling downy mildew is required.
Plant induced resistance is a method to activate self resistance in plants by giving inducer materials which trigger the plant defense mechanism. Therefore plants become more resistant against subsequent pathogen infection (Conrath et al., 2002;Agrios, 2005;Faoro et al., 2008). External inducing materials (elicitors) can be biological, chemical and physical agents (Agrios, 2005;Widiastuti et al., 2013). According to Faoro et al. (2008), resistance induction offers several important advantages, such as it is not based on a modification of plant genomes, relatively cheap, it has no environmental risks; and effective against various plant pathogens.
Research using non-fungicide chemicals as inducers of plant diseases in Indonesia has not been much conducted (Habibullah et al., 2018), especially on maize. In previous research, Habibullah et al. (2018) reported that maize seed dipping treatment by nonfungicide chemicals such as benzoic acid, sodium benzoic, salicylic acid or saccharin induced early response of plant defense by forming reactive oxygen species (ROS) and the presence of mycelial lignification inside the plant tissues. Continuing the research, this study was conducted to observe those non-fungicide chemicals which were potential to suppress downy mildew of maize.

Preparation and Inoculation
Maize seeds (sweet corn variety) from local seed producers without fungicide application were used in this research. Seeds were soaked in benzoic acid, sodium benzoate, salicylic acid, thiamine, saccharin and aspirin, each with concentrations of 2000 ppm distilled water for 60 minutes. Seeds were planted in pots (5 kg of soil/pot) with 5 replications and 5 seeds/pot. The positive (+) control was seeds immersed in distilled water and inoculated, while the negative control (-) was seeds immersed in distilled water without inoculation. Inoculation was carried out at 10 days after planting following method of Yuniasih and Sumardiyono (2014). Inoculum source was downy mildew diseased leaves collected from Klaten, Central Java. The leaves were cut approximately 2×3 cm and inserted into the middle of the youngest tested leaves. Inoculation was conducted at around 5 p.m. To maintain moisture, the base of the plant was covered by wet cotton and the whole plants was covered with plastic bag over night.

Observation of Disease Incidence, Severity and Development of Downy Mildew of Maize Plants
Observation of disease incidence (%) and severity (%) of downy mildew was conducted every week for 5 weeks after pathogen inoculation. Disease incidence was calculated using the following equation:

Observation of Lignification on Maize Leaf Tissue
Observation of leaf tissue lignification was carried out at the 3 rd week after inoculation, following the Sass method (1958) with slight modification. Maize leaves were cleaned, cut approximately 1 cm, then clamped in the middle of cassava cork and thinly sliced transversely using a razor. The leaf slices were soaked with 0.05% phloroglucinol for 15 minutes, then soaked in 25% HCl solution for 30 seconds. Leaf slices were placed on a glass slide, covered by a cover glass and observed microscopically. Leaf tissue showed purple-red discoloration indicated tissue lignification (Sass, 1958

Observation of Maize Plant Growth
Parameter of plant growth measured in this research was plant height, number of leaves, and root length of the plants. Observation of plant height and number of leaves was done every week during 6-week planting. Plant height measurements were carried out by measuring the length from the base of trunk to the longest leaf tip. Observation of root length was done at the 6 th week after the planting.

Chlorophyll Content of Maize Leaves
Observation of chlorophyll content was carried out at 5 th week after inoculation. Observation was made on each treatment represented with 3 samples as replication. Leaf samples used were 3 top or young leaves of every single plant. The method referred to Arnon (1949). Fresh leaves were weighed as much as 1 g. Acetone 80% as much as 20 ml was added to leaf samples and crushed using a mortar. The crude extract was filtered using filter paper and then put into a test tube. The solution was poured into a spectrophotometer cuvette up to boundary line and measured the absorbance value. Pure acetone solution was used as a standard solution. Observations were made at wavelengths of 645 nm and 663 nm. The absorbance value obtained was used to calculate the chlorophyll content using the formula according to Arnon (1949): Total chlorophyll = 20,2 A645 + 8,02 A663 mgg -

Dry Weight
Dry weight was measured at 6 th week after planting. Plants were dried in an oven at 60 ºC for 4 days.

Data Analysis
Agronomic observations of maize plants including height, number of leaves, root length and dry weight were analyzed using ANOVA (analysis of variance) at 5% significance level followed by Duncan New Multiple Range Test (DNMRT) at the same level of significance.

Disease Incidence, Severity, and Progression on Maize Downy Mildew After Treatment
The result showed that the tested chemicals reduced the percentage of disease incidence ( Figure 1) and disease severity of downy mildew (Figure 2) of maize plants. Observation at 5 weeks after inoculation proved that disease incidence of plants treated by benzoic acid was 20%, sodium benzoate and saccharin were 30%, aspirin was 40%, and salicylic acid and thiamine were 50%, while inoculated plants (positive control) was 100%. Furthermore, the disease severity of plants treated by benzoic acid was 20%, sodium benzoic was 30%, saccharin was 25%, aspirin was 27.5%, salicylic acid was 35% and thiamine was 40%, whereas in plants that were only inoculated (positive control) was 90%.
The result showed that benzoic acid gave the best result in reducing disease incidence and severity. Benzoic acid suppressed disease incidence and disease severity up to 80% compared to the positive control. Categories of plant resistance is performed in Table 3. Data showed that chemicals increased plant response against downy mildew for moderately  (Table 4).
On data average, all treatments showed slower disease progress compared to positive control. Those data demonstrated that benzoic acid and sodium benzoate treatment possessed potential ability in controlling maize downy mildew. This occurs because of the ability of these chemicals to induce plant resistance so that it is more resistant to pathogen attack. This result is in accordance with Habibullah et al. (2018) which showed that chemicals in the form of sodium benzoate and benzoic acid had ability to increase the resistance of maize plants to P. maydis attack. In addition, research by Fadel et al. (2006) also showed that benzoate compounds could affect the attack of Ustilazo maydis on maize.

Lignification of Maize Leaf Tissue
Microscopic qualitative observation (Figure 3) showed that lignification occurred in the tissue around the stomata of maize plants in all inoculated plants treated with chemicals, but it was not found in uninoculated plants. Lignification was indicated by the presence of purplish-red in the leaf tissue dyed with phloroglucinol (Sass, 1958). Lignification surrounding the stomatal area in inoculated plants without any chemical treatments (positive control) showed a general response of maize plants as their  defense mechanism agints pathogen infection. He et al. (2002) showed that cell wall lignification in pathogen-infected tissue was one of the plant defense systems. This result resembled to Asmawati (2016), which showed lignification in the surrounding of stomata of the maize leaves induced resistance with Trichoderma spp. The presence of lignification in the tissues around leaf stomata showed that plants activated their defense mechanism after treated with chemical inducer or pathogen inoculation. Vance et al. (1980) stated that increasing level of lignin inhibited physical penetration and invasion of pathogens, inhibited toxins spreading and enzymes released by pathogens, as well as inhibited nutrients supply needed by pathogens. Previously, Habibullah et al. (2018) reported that the same chemical treatment produced reactive oxygen species (ROS) in leaf tissue as plants early response and mycelial lignification of pathogen in leaf tissues. Stomata lignification in this study showed that the chemical treatments possibly induced maize resistance and then suppressed the incidence and severity of the disease. Some previous studies also showed the effect of chemicals on suppressing disease in plants. Fadel et al. (2006) reported that treatment of salicylic acid, sodium benzoate, and potassium monophosphate could suppress swollen rot disease of maize plants. In that research, severity of fruit swelling in plants treated with sodium salicylic acid was 10.12%, benzoate was 6.34%, and potassium monophosphate was 5.83%.

Growth of Maize Plants
Result showed that chemical treated plants had better growth compared to positive control and were not significantly different from negative control (Table 5). Taken together with previous data that  chemical treatment reduced disease incidence and severity, it was assumed that plants had better endurance in their growth stage, therefore plant height and the number of leaves were higher than the positive control. On root length, there were no significant difference among treatments, but salicylic acid treated plant showed the longest root (Table 5). Some reports showed that certain chemical treatment influenced plant physiology, such as Hayat et al. (2005) which informed that salicylic acid regulated the balance of auxin, cytokinin, and ABA in plant tissues, induced the proliferation and expansion of plant cells, increased the absorption and transportation of nutrients, increased photosynthesis, thereby increased plant growth.

Chlorophyll Content and Dry Weights of Maize Plants
Observations of chlorophyll content (Table 5) showed that the highest value was gained in negative control at 0.85 mg/g, followed by plants treated with sodium benzoate, and other chemicals. The lowest chlorophyll content was shown in the positive control at 0.35 mg/g. Those data related to previous observation that disease incidence and severity was lower after chemical treatments, indicated that plants might compensate the pathogen infection process. Therefore their physiological process was not much disrupted including the clorophyl production. As the consequence, chemical treated plants would grow better. Plant resistance to P. maydis caused lower leaf chlorosis or the chlorophyll content in the leaf was well maintained, in accordance with the research of Agustamia (2016) and Asmawati (2016) which showed that plants that were more resistant to P. maydis had a higher chlorophyll content than susceptible plants.
The highest dry weight was obtained by negative control at 3.06 g, followed by plant treated with 3 g of benzoic acid, 2.67 g of sodium benzoate, 2.63 g of saccharin, and 2.6 g of salicylic acid. All of these treatments were not significantly different from negative control. The lowest dry weight was found in positive control and significantly different from all treatments. The low dry weight of positive control was probably caused by the lack of chlorophyll content which resulted in inhibition of plant growth rate due to decreasing photosynthate. This is in line with the determination of Habibullah & Suhendra (2020) which showed that maize plants that were induced resistance showed higher dry weight than positive control.

CONCLUSION
The benzoic acid treatment of 2000 ppm gave the lowest disease incidence, intensity and progression compared to other treatments and positive control. Plants resistance category was maintained in moderately resistant up to 5 th week after planting. Plant growth, dry weight, and chlorophyll values were not significantly different from negative control. Therefore benzoic acid had the highest potential as a resistance inducer chemical of maize to downy mildew for further research. Note: The numbers in the same column followed by the same letters indicate no significant difference based on the DNMRT test with a 95% confidence level.