Genetic response of hybrid maize line to downy mildew caused by Peronosclerospora spp. infection

Maize is the second staple food commodity in Indonesia. Apart from being the main source of carbohydrates and protein, the production of maize continues to increase along with the escalation of population growth and animal feed requirements in the last few years. The potential to increase the national production of maize is still feasible because of the yield gap between the potential yields of new superior varieties and the level of yields obtained by farmers. The yield gap caused by biotic stress in maize is mainly caused by pathogens such as downy mildew due to Peronosclerospora spp. Downy mildew distribution is sporadic that can infect a wide area. In Indonesia, it spreads widely and significantly reduces yields in the areas of maize production centres in East Java, Central Java, South Sulawesi, North Sulawesi, Gorontalo, Lampung, and Sumatera. These obstacles can be overcome by integrated pest and disease control technology. One strategy is to discover downy mildew-resistant varieties that can be combined with other control treatments. The phenomenon of resistance to downy mildew infection of several hybrid maize strains began to be detected in the vegetative growth phase, with symptoms beginning at 14 days after planting (DAP), increasing with plant age, and reaching its peak after 28-35 DAP and then the symptoms will gradually disappear until no infection after 42 HST. This study analyses the resistant maize varieties from 2020 to 2022. The data showed the susceptible comparison variety (Anoman) was infected with 88.94% to 100%. In 2020, BMD73 showed a resistant reaction to Philippinensis species, other strains were classified as susceptible in P. maydis except BMD 76. All strains showed highly susceptible and susceptible reactions. In 2021, in P. philippinensis all strains FCP10-FCP16 showed a resistant reaction, but line FCP10-FCP16 had a resistance response that was classified as moderately resistant to P. maydis. The incidence of genetic response in 2022, except BMD86, strains BMD81-BMD85 were classified as resistant, as well as in the P. maydis endemic area all strains showed the same reaction.


Introduction
One of the constraints to maize production in Indonesia is downy mildew caused by the pathogen Peronosclerospora spp.Improvements in downy mildew resistance have been widely reported, but highly resistant varieties have not yet been obtained.Current high-yielding varieties emphasize productivity, environmental stress tolerance, better nutritional quality, and stay-green biomass for animal feed purposes.The combination of these traits has proven to increase yield but has not been able to overcome downy mildew problems.Downy mildew (Peronosclerospora spp.) is a serious constraint in maize production centers in Indonesia.Downy mildew is found and develops in the field with the main host plants being maize 1230 (2023) 012091 IOP Publishing doi:10.1088/1755-1315/1230/1/012091 2 and sorghum [1,2].Research results [3,4] showed that three species of downy mildew in Indonesia have been found, namely P. phlippinensis.P. maydis and P. sorghi.P. maydis was found in Java and P. philippinensis spread in Sulawesi.
The disease became widespread after the national expansion program and efforts to increase maize production, due to the sporadic, fast-spreading dynamics of the infection and the limited use of resistant new improved varieties at the farm level.Downy mildew has also recently been reported to infect new and improved varieties in the early growth phase and has the potential to significantly reduce maize yields on a national scale.The presence of early sources of inoculum, due to the planting of susceptible maize varieties, and non-consistent planting patterns in each maize-growing region means that downy mildew is always present and remains a threat to meeting Indonesia's production targets.Early infection, in susceptible varieties, can cause yield losses of around 90% [5,6].
The use of resistant varieties is one of the most effective components in the control strategy of major maize diseases [7][8][9].The usage of one variety will lead to the emergence of new races of downy mildew that are more virulent, so that resistant varieties usually after planting a few seasons will experience a decrease in resistance [10,11].Research results of [12] reported that from the test of maize germplasm resistance to downy mildew (Peronosclerospora spp.) collected in the last 5 to 6 years from various regions in Indonesia.Only about 0.5-1 percent was identified that showed resistance to downy mildew.This situation illustrates that the availability of downy mildew-resistant elders is relatively rare so efforts to find high-yield potential varieties based on resistant gene sources are continuously needed so that the availability of resistant varieties can always be available when needed by farmers.
Other important lessons are the need to understand the dynamic phase of infection rates in the field, distribution data, and peak infection related to plant age.These bionomic characters are expected to be a comprehensive control component in the concept of integrated control of downy mildew in maize.This paper will discuss the genetic response, and infection dynamics, of downy mildew stress in Filial 1 (F1) hybrid maize strains in the last 3 years, namely 2020, 2021, and 2022 in two distribution areas of the causal species, namely P. philippinensis and P. maydis.

Research Methodology
Research to test the dynamics and resistance to downy mildew was conducted in endemic areas of P. philippinensis (Bajeng, Gowa, South Sulawesi) and P. maydis (Kediri, East Java) [13,14].Activities in the region of P. philippinensis species were carried out in late May to early September from 2020, 2021 and 2022, at IP2TP Bajeng, Gowa, South Sulawesi and for P. maydis species were carried out in Kandangan, Kediri, East Java.The number of candidate varieties tested was 6-7 Filial 1 (F1) maize lines from each year, namely in 2020 (BMD71, BMD72, BMD73, BMD74, BMD75, BMD76), 2021 (FCP10, FCP11, FCP12, FCP13, FCP14, FCP15, FCP16), and 2022 (BMD81, BMD82, BMD83, BMD84, BMD85, BMD86).All of these F1 test strains are the result of breeding conducted by several seed producers in Indonesia, as well as 2 comparison varieties which represent the Anoman variety as a susceptible comparison and Pertiwi-6 and Pioneer-6 (P6) as a resistant comparison to downy mildew (Peronosclerospora spp.).The determination of the comparison varieties was carried out with the following considerations: 1. Anoman variety: the most susceptible variety to downy mildew (Peronosclerospora spp).2. Pertiwi 6: is a commercial variety resistant to downy mildew (Peronosclerospora spp).The experiment was arranged in a Randomized Block Design with 3 replications.The susceptible comparison variety was Anoman, and the resistant comparison varieties were Pertiwi-6, and P36.

Inoculum source preparation
Inoculum source or spreader plants (Anoman variety) were planted three rows around the perimeter of the test plot and 3 rows between each replicate.Ten days after planted (DAP), maize inoculated with the conidia suspension of P. philippinensis and P. maydis.The suspension spread in the dawn, around 5 AM when there was still dew in the cropping area.

Test material
The test materials (candidate varieties), planted in 4 rows (5 x 2.8 m), spacing (70 x 20 cm), 3 replications, when the source of inoculum (spreader plants) around the experimental plot was infected with downy mildew > 70%.Two seeds were planted in each hole and Carbofuran 3G was applied to prevent ants or leaf-eating pests.Thinning of plants was done at 7 DAP by leaving one plant per hole.The first fertilization was given at the age of 10 DAP with Urea and Phonska at 150 kg/ha and 200 kg/ha, respectively.The second fertilization was done at the age of 30 DAP by giving Urea 150 kg/ha.Every 3 test materials were planted with a susceptible variety (Anoman variety) and a resistant variety (Pertiwi-6).

Research variable
Infection dynamics calculated based on the intensity of downy mildew attack, is carried out based on the incubation period, namely the vulnerable time between initial penetration until the onset of downy mildew symptoms at 14, 21, 28, 35, 42 DAP, as follows: The percentage of infestation was calculated using the formula:  = (/)  100% I = Percentage of downy mildew infestation A = Number of downy mildew-infested plants B = Number of plants observed in each strain The research data is continued with analysis using variance analysis, followed by LSD (Least Significance Different) test.Criteria for determining the resistance of strains to downy mildew are taken from the last observation of 42 DAP with reference to the Committee for the Release of Varieties of Food Crops of the Ministry of Agriculture (PPVTP 2018) as follows: a) Very Resistant: disease incidence 0-5% b) Resistant: disease incidence >5 -20% c) Moderately Resistant: disease incidence >21 -40% d) Susceptible: disease incidence >41 -60% e) Highly susceptible: disease incidence >60

Downy mildew infection dynamics (P. philippinensis and P. maydis)
Downy mildew in maize is caused by a pathogenic fungus belonging to the genus Peronosclerospora spp.This fungus belongs to the family Peronosporaceae, with the genus Peronosclerospora consists of seven species with the main host plants being maize and sorghum [15,16].In Indonesia, three species are reported, namely.P. maydis, P. philippinensis, and P. sorghi, but there are two species that dominantly attack maize crops, namely P. maydis and P. philippinensis [15,[17][18][19].The species P. maydis is found in the Java region and P. philippinensis spreads in the Sulawesi region [14,20,21].The results of [22] showed that in Berastagi District, North Sumatra province, P. sorghi species were also found to infect maize plants.Downy mildew conidia germinate at midnight and infects after germinating in guttation dew droplets at the growing point of the leaf base of maize plants.The spores of P. maydis are rounded [14], (17-23x27-39 nm) in diameter, and P. philippinensis are slightly oval, (21x27-38 nm) in diameter [23].The two species have been reported to have different levels of virulence when infecting maize plants [24].
Research conducted since 2020, 2021 and 2022 in the endemic areas of P. philippinensis and P. maydis, showed that the distribution of inoculum source plants (spreader plants) planted around the strains tested for resistance to downy mildew, the intensity of downy mildew reached around 80%-98%.The high availability of inoculum source plants caused the test strains in the treatment plots to be evenly infected naturally and there was no chance of the test strains being protected from the spread of downy mildew conidia infection (escape).
Visual symptoms typical of downy mildew in the study area endemic to P. philippinensis and P. maydis do not have characteristic symptoms that distinguish them.It is found with a yellow, whitish chlorotic color line following the direction of the leaf bone, infecting from the base of the petals, spreading to the tip of the leaf, the leaves become stiff and erect, and the plant becomes stunted.The underside of infected leaves is characterized by the presence of white powdery powder, which is the conidia of downy mildew.The characteristic symptoms of downy mildew are as stated by (21,22).In the field, it was also found that in resistant varieties, plants did not show symptoms of leaf discoloration, plants grew normally (Figure 1), while in susceptible varieties, downy mildew caused leaf discoloration and plants became stunted and some individuals in the population dried up and died (Figure 2).This pathogen infects the growing point, systemically, extending to all parts of the plant.All early symptoms of downy mildew are found at the base of the stem or growing point, at the base of the stem of the maize plant in the early morning there is guttation so that the conidia that settle on the base of the stem can reproduce perfect sprout tubes and have a high chance of making initial penetration on the leaf surface.On the other hand, for conidia that settle on other parts of the leaf surface, the dew water dries quickly so that the conidia that settle when germinating will experience the death of the sprout tube before reaching the stomata and do not cause infection, so there are never any initial symptoms found on other leaf surfaces.
Observations during the years; 2020, 2021, and 2022 (figure 3 and 4), both in the endemic areas of the species that cause P. philippinensis and P. maydis indicated that all infection events were sourced from the spreader plants (inoculum sources) planted on the perimeter of the research activities and there were no infection events because of seed transmission.[27] state that downy mildew can only be transmitted through the air and cannot be transmitted through seeds.In all test strains Filial 1 (F1) showed early infection with low attack found at the age of maize plants, 14 DAP.Some symptoms were limited to the petals at the base of the leaves and the symptoms had not spread systemically to all parts of the plant.The lower intensity of infected plants in the early growth period is due to the incubation period or the time span between the initial penetration of conidia and the onset of symptoms, around 7 to 10 days.The incidence of infection symptoms at the age of 14 DAP indicates that the initial penetration of conidia in the field has occurred as soon as the plants grow around 5 or 7 DAP, and the symptoms then appear after the plants are 14 DAP.In figure 3 and 4, 21 DAP, the dynamics of infection began to increase following the increase in plant age.The increase in the area and number of leaves as a medium for conidia, causes conidia reproduction in the field to also increase so that the increase in infection intensity is directly proportional to the increase in plant age.All Filial 1 (F1) showed different infection dynamics, but the population of infected plants increased with plant age.The peak infection rate of downy mildew seems to occur after the plants are 28-35 DAP (Figure 3 and 4), then the increase in intensity will decrease, inversely proportional to the increase in plant age.The incidence of additional infected plants is different from each test strain, depending on the genetic response of each F1 strain tested.The incidence in the field was also corrected in the susceptible comparison variety (Anoman) which reached 98%-100% infection intensity and the resistant comparison varieties Pertiwi-6 and P 36 whose intensity remained low (5.65%-6.33%)respectively and were classified as resistant to downy mildew (P.philippinensis and P. maydis).
The high intensity in the susceptible comparison varieties indicates that the reproduction of downy mildew conidia in the field is optimal and the absence of strains that show a low infection reaction is only due to escape from infection with the pathogens P. philipinensis and P. maydis.The infected plant tissue vessels can be filled by invading pathogen masses, and this occurs because of the positive response between the host and the pathogen which causes blockage of plant tissue vessels [28] situation causes susceptible host plants to be unable to grow normally and causes some individuals in a population to usually dry up and die (fFigure 2).
In two endemic areas, the distribution of species that cause downy mildew disease (P.philippinensis and P. maydis).The reduction of infection started after 42 DAP (≤1%) or in some treatment plots, there were no newly infected plants.The decrease of infected plants is also due to the thickening of the leaf epidermis layer in the growing-plants, causing a reduction in the susceptibility to downy mildew.This three years research provide important information, that the dynamics of infection distribution tend to be sporadic in the vegetative growth phase.The characteristics of its distribution and short life cycle of about 7-10 days, cause the potential for sporadic attacks.High yield loss is caused by the Peronosclerospora spp.because the pathogen started in affected the plants in vegetative phase and utilized plant nutrient caused the stunted.Maize plants that are infected early in the vegetative phase usually do not produce cobs or the number of seeds is less in each cob.Similarly, the size of the seeds produced in infected plants was smaller than the production of healthy plants [29,30].Therefore, in relation to the intensity of distribution dynamics, downy mildew control will only be effective in the early vegetative growth phase through seed treatment or foliar application of fungicides.

Resistance response of Filial 1 (F1) strains to downy mildew caused by P. philippinensis and P. maydis
In general, the cause of the disease is determined by three disease components: conidia density, rainfall, and length of irradiation.The average wind speed and plant position also play a role in the disease distribution.The genetic factor influences plant severity to downy mildew [31,32].The intensity of the disease to attack maize in this research presented the different resistance reactions (tables 1, 2 and 3).
Data in 2020 (table 1) indicate that all the line/genotype showed highly susceptible to P. philppinensis, and P. maydis except BMD73.The strains classified as susceptible and very susceptible are BMD71, BMD72, BMD75, in a situation where the susceptible comparator (Anoman) was infected > 98.17% and the resistant comparator (P 36) was infected 5.47%.In 2021 (table 2), the result indicate that the line strains FCP10-FCP16 showed an infection reaction to downy mildew (P.philippinensis) 10-15% or significantly lower than the susceptible comparator (Anoman variety) which was infected with 97.94%.This situation was also strengthen by the intensity of infection in the resistant comparator (variety P 36) which was only infected by 6.40%.In the P. maydis endemic area, genotype FCP10-FCP16 were infected in the range of 20.82%-35.06% or very significantly lower than the susceptible comparator (Anoman variety) which was infected 88.94%.When referring to the resistance criteria of PPVTP (2018), these strains in infection caused by P. philippinensis species are classified as resistant and infection caused by P. maydis species is classified as moderately resistant.The very high infection intensity in the susceptible comparison of 88.94%-97.94%indicates that the distribution of conidia in the research activity area is normal so that all genetic reactions expressed by each test strain are optimized according to their resistance traits and there are no strains that show resistant reactions only because they are protected from the distribution of downy mildew conidia in the field.The virulence level of the pathogen P. maydis in the Kediri endemic area, Java Island is higher than the virulence of the dominant P. philipinensis species in Gowa, South Sulawesi [33].The active resistance mechanism of a variety is a structural and biochemical response aimed at protecting host tissues from pathogen colonization.It starts with a signal from the pathogen, then induces phosphorylation that is graded according to the genetic response of the invaded host, then shows symptoms of a different resistance response from each host.The lower intensity reaction, in strains BMD71-BMD76, and FCP10-FCP 16 were because these strains have resistant genes, which can limit the initial infection of downy mildew pathogens (P.philippinensis and P. maydis) or downy mildew conidia can infect but do not develop in plant cell tissue so the development of downy mildew in the treatment plots is lower compared to other strains that are more susceptible.
The initial penetration by a pathogen begins with the release of phytotoxins, then the plant reacts, defending itself with the release of phytoalexins.Phytoalexin inhibition is not dominant, resulting in the pathogen being able to develop well and causing the plant to become susceptible [34,35].In test strains that are classified as susceptible, the toxin compatibility of P. philippinensis with its host is thought to be high, allowing downy mildew conidia mycelia to develop well in plant cell tissue and optimally infected plants.
The response of BMD 81-BMD 85 to P. philippinensis infection was resistant trait (5-10%) but susceptible in genotype BMD-86 (42.48%).In the infection of P. maydis, the strains BMD81-BMD85 were infected with 4-16% or significantly lower than the susceptible comparison variety (Anoman) whose infection reached 93.47%.The relationship between the level of resistance of a variety and the level of virulence of the pathogen, as well as the ability of the pathogen to produce toxins, can lead to optimal early infection of plants in the field.Non-dominant phytoalexin inhibition results in the pathogen being able to develop well and cause the plant to become susceptible [36].In test strains that are classified as susceptible, toxin compatibility with the host is thought to be high, allowing downy mildew conidia mycelia to develop well in plant cell tissue.These events cause differences in the resistance properties of several varieties from susceptible, moderately susceptible, and resistant varieties.The use of resistant varieties in downy mildew control is the most effective component [26].Resistant varieties can suppress conidia production, reduce initial inoculum sources, and slow down the sporadic nature of downy mildew transmission.However, resistant varieties should not be planted continuously, because they can increase the selection pressure of P. maydis and P. philippinensis races that allow the development of new and dominant and more virulent races, so that the resistance of a maize variety to downy mildew can be broken.[33] reported that varieties such as Lagaligo composite maize, Bima 3 Bantimurung and hybrid maize Bima 15 Sayang, Bima 20 URI, which were initially released, were classified as resistant to downy mildew but have decreased their resistance to become somewhat resistant.
Another thing that can be a concern in stabilizing the sustainability of a variety's resistance to pathogens is that in every formation of Filial 1 (F1) generation hybrid maize, the purity and recombination process of elders from fixed genetic sources must be necessary.The obstacle to the formation of downy mildew-resistant varieties is that generally the source of resistance is controlled by many genes, and the downy mildew pathogen itself is very easy to form new physiological races to adapt [37].Therefore, it cannot be ignored that the maize germplasm collection program or the management of the "Genetic Bank" as a source of downy mildew resistance genes must still be carried out.The evaluation of 300 accessions of germplasm resistance to downy mildew over two growing seasons, only about 0.5% of accessions were classified as resistant [38].The discovery of sustainable resistant varieties can be one component of integrated disease control.The use of resistant varieties can be combined with other control components to be more effective in suppressing downy mildew in maize development centers in Indonesia.

Conclusions
Downy mildew infection caused by the species P. philippinensis and P. maydis, began to appear in the vegetative growth phase, early localized symptoms on the growth point at the base of the leaves at the age of 14 DAP, increased following the development of plant age, reaching a peak after 28-35 DAP, then very low infection or no infection after 42 DAP.
In the situation of susceptible comparison varieties (Anoman), 88.94% to 100% infection.Activities in 2020, BMD73-75 showed a resistant and moderately resistant reaction to P. philippinensis species, other strains were classified as susceptible, in P. maydis species except BMD 76, all strains showed very susceptible and susceptible reactions.The incidence of infection in 2021, in the P. philippinensis region, all strains FCP10-FCP16, showed a resistant reaction, while in the P. maydis region, strains FCP10-FCP16 had a moderately resistance response that was classified as resistant.In 2022, except BMD86, strains BMD81-BMD85 had a resistant genetic response, as well as in the P. maydis endemic area all strains showed resistant and highly resistant reactions to downy mildew.

Suggestion
From the dynamics of infection, it can be explained that downy mildew will only be effectively controlled in the early growth phase either by seed treatment or suspense treatment on leaves in the early vegetative growth stage.