Pollen viability, physiology, and production of maize plants exposed to pyraclostrobin + epoxiconazole

    Pollen viability, physiology, and production of maize plants exposed to pyraclostrobin + epoxiconazole Verˆonica Barbosa Junqueira, Alan Carlos Costa, Tatiana Boff, Caroline M¨uller, Maria Andr´eia Corrˆea Mendonc¸a, Priscila Ferreira Batista PII: DOI: Reference:

S0048-3575(16)30109-2 doi: 10.1016/j.pestbp.2016.09.007 YPEST 3991

To appear in: Received date: Revised date: Accepted date:

8 July 2016 20 September 2016 23 September 2016

Please cite this article as: Verˆ onica Barbosa Junqueira, Alan Carlos Costa, Tatiana Boff, Caroline M¨ uller, Maria Andr´eia Corrˆea Mendon¸ca, Priscila Ferreira Batista, Pollen viability, physiology, and production of maize plants exposed to pyraclostrobin + epoxiconazole, (2016), doi: 10.1016/j.pestbp.2016.09.007

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Pollen viability, physiology, and production of maize plants exposed to pyraclostrobin + epoxiconazole

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a

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Maria Andréia Corrêa Mendonçac, Priscila Ferreira Batistaa

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Verônica Barbosa Junqueiraa, Alan Carlos Costaa*, Tatiana Boffb, Caroline Müllera,

Laboratório de Ecofisiologia e Produtividade Vegetal, Instituto Federal de Educação,

Ciência e Tecnologia Goiano – Campus Rio Verde, Caixa Postal 66, 75901-970, Rio Verde,

GO,

Brazil.

([email protected],

[email protected],

Instituto Federal de Educação, Ciência e Tecnologia do Triângulo Mineiro – Campus

Uberlândia,

Caixa

Postal

([email protected]) c

1020,

38400-970,

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b

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[email protected], [email protected])

Uberlândia,

MG,

Brazil.

Laboratório de Biotecnologia, Instituto Federal de Educação, Ciência e Tecnologia

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Goiano – Campus Rio Verde, Caixa Postal 66, 75901-970, Rio Verde, GO, Brazil.

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([email protected])

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*Corresponding author (A.C. Costa): Instituto Federal Goiano – Campus de Rio Verde Av. Sul Goiânia, Zona Rural, s/n

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Rio Verde – GO, Brasil 75901-970 Tel #: 55 64 3620-5617; Fax #: 55 64 3620-5640 E-mail address: [email protected]

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ABSTRACT

The use of fungicides in maize has been more frequent due to an increase in the

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incidence of diseases and also the possible physiological benefits that some of these products may cause. However, some of these products (e.g., strobilurins and triazoles)

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may interfere with physiological processes and the formation of reproductive organs. Therefore, the effect of these products on plants at different developmental stages needs to be better understood to reduce losses and maximize production. The effect of the

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fungicide pyraclostrobin + epoxiconazole (P + E) was evaluated at different growth stages in meiosis, pollen grain viability and germination, physiology, and production of maize plants in the absence of disease. An experiment was carried out with the hybrid

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DKB390 PROII and the application of pyraclostrobin + epoxiconazole at the recommended dose and an untreated control at 3 different timings (S1 - V10; S2 - V14;

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S3 - R1) with 5 replications. Gas exchange, chlorophyll fluorescence, pollen viability and germination, as well as the hundred-grain weight were evaluated. Anthers were

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collected from plants of S1 for cytogenetic analysis. The fungicide pyraclostrobin + epoxiconazole reduced the viability of pollen grains (1.4%), but this was not enough to

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reduce production. Moreover, no differences were observed in any of the other parameters analyzed, suggesting that P+E at the recommended dose and the tested

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stages does not cause toxic effects.

Keywords: chemical control; corn; strobilurin; triazole; Zea mays.

1 INTRODUCTION

Maize (Zea mays L.) is one of the major crops of the world and is used for human and animal consumption as well as a raw material for products as rubber, plastic, biodiesel, and fabric [1]. In the 2014/2015 crop, more than one billion ton of maize was produced in the world, and the largest producer was the United States (261.1 million tons [35.8 %]), followed by China (21.4 %), and Brazil (8.4 %) [2]. Z. mays is a C4 plant that has high yield potential. However, inadequate pest management can limit the production capacity in the field. Therefore, maize productivity (on average 5 ton.ha-1 in

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Brazil) has been considerably below its potential, which reaches 14 ton.ha-1 in areas with more improved production systems [3]. One of the main factors contributing to the reduction of the maize crop

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productivity is the incidence of diseases combined with improper management [4]. The

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increase in maize diseases in Brazil is a consequence of the crop production system

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evolution, which confers modifications as expansion of planting dates as well as the use of no-till systems and increased irrigation [5]. For this reason, the use of fungicides has increased in several countries in order to control disease and maintain productivity [6]. Fungicides most commonly used for foliar application in maize belong to the

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triazole and strobilurin chemical groups. Today in Brazil, there are 62 commercial products registered for maize disease control, 70% of those having triazole and

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strobilurin individually or combined [7]. The triazoles inhibit ergosterol synthesis in fungi [8, 9], which has a similar function to the phytosterols [10]. On the other hand, strobilurins block the complex III at the mitochondrial electron transport chain [11, 12].

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When applied to plants, these fungicides may create effects other than pathogen control,

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causing negative [13] or beneficial [6] changes in plants. One factor that should be considered when applying fungicides to maize plants

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is the possibility of the phytotoxic effects of these products. According to Petit et al. [14], the use of fungicides can result in disruption of the development of reproductive organs and reduction of plant growth. Triazoles, for example, may cause changes in

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chloroplast electron transport and phytosterols synthesis, as observed in Gallium aparine [13], which may be related to the pollen grain inviability [15]. Anomalies caused during meiosis may also reduce the viability of pollen grains of various species [16]. However, there is little information regarding the action of fungicides in cell division in maize plants. Similarly, strobilurins may reduce the respiration rate in plants, which is related to its action mechanism in fungi [12]. Another possibility is that fungicides can promote growth, which is also known as a ‘physiological effect’ [17]. These beneficial effects are attributed to fungicides that improve the physiological traits such as photosynthesis, antioxidant system, nitrogen metabolism, and grain yield even in plants in the absence of illness [6, 18]. Strobilurins are known to promote ‘physiological effects’ in several crops, as observed by the potential increase in productivity in maize plants by Wise and Mueller [19]. Triazoles have been described

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by promoting responses to pre-inducing resistance to abiotic stresses, such as drought [20]. These physiological effects have enticed producers to invest in fungicides even

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in crops with low or no disease pressure since they hope for possible increases in yield.

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However, Paul et al. [21] suggest that foliar application of fungicides in maize rarely

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results in economic benefit when the severity of the diseases is low and the expected productivity is high.

In order to decide on the application of a fungicide in maize, the following should all be taken into account: stage of development, environmental conditions,

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presence of inoculum, susceptibility of the cultivar, and disease severity [22]. It is also important to emphasize that the economic return depends not only on the increase in

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production but also on the cost of the application of the products, which involves the price of the commercial product and its application in addition to the market price of maize [21, 23].

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The best timing of fungicide application in maize needs to be better understood

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in order to maximize grain yield and economic benefits; this should consider disease control but also the possible physiological benefits or phytotoxic effects. We tested the

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hypothesis that the fungicide pyraclostrobin + epoxiconazole at a commercial dose can affect the reproductive development of maize plants, and these toxic effects can be minimized if the most sensitive phenological stages are known. Therefore, this study

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aimed to evaluate the effect of the fungicide pyraclostrobin + epoxiconazole applied at the recommended doses on different stages of development of pollen grains of maize through physiological, cytogenetic, and production analysis.

2 MATERIAL AND METHODS

2.1 Plant Material and Experimental Conditions The experiment was performed in a climatized greenhouse at the Laboratory of Ecophysiology and Plant Productivity at the Instituto Federal Goiano, Campus Rio Verde, Goiás, Brazil. Hybrid maize seeds (DKB390 PRO II, Monsanto, Brazil) were sown in plastic pots containing 18 L of substrate. The substrate was prepared from a mixture of Red Latosol (LVdf) soil and sand in the ratio 2:1, which was fertilized with nutrient solution according to the soil chemical analysis and the recommendation for

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high maize yield [24]. The experiment was carried out from October 2015 to February 2016. Initially, ten seeds were sown per pot, and after germination only one plant was kept per pot, corresponding to one experimental unit. All plants were kept at field

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capacity and absent of pests and diseases throughout the cycle.

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Treatments consisted of the fungicide pyraclostrobin + epoxiconazole (Opera® BASF Crop Protection, Limburgerhof, Germany) applied at 0.133 and 0.500 kg a.i. ha-1,

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respectively (0.75 L of commercial product ha-1), and an untreated control in the following growth stages: V10 (S1), which corresponds to 10 unfolded leaves; V14 (S2), which corresponds to 14 unfolded leaves or end of leaf development; and R1 (S3),

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which corresponds to the flowering (or opening of anthers). The fungicide application was carried out using a CO2-charged hand boom sprayer equipped with 4 Tee Jet

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nozzles, which delivered 200 L ha−1, as recommended for disease control on maize. Sprinkling was performed directly over the plants by keeping the bar at 0.4 m high from the top of the plants. The experimental design was randomized blocks in a factorial

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scheme 2 (doses) x 3 (times) with five replicates.

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2.2 Physiological traits

2.2.1 Gas exchange Gas exchange from maize plants were measured five days after the fungicide

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application (DAA) to determine the net photosynthetic rate (A, μmol CO2 m-2 s-1), stomatal conductance (gs, mol H2O m-2 s-1), transpiration rate (E, mmol H2O m-2 s-1), the ratio between internal and external CO2 concentration (Ci/Ca), and the dark respiration (RD, μmol CO2 m-2 s-1). The parameters were measured using an infrared gas analyzer (IRGA, model LI-6400XTR, LI-COR, Lincoln, Nebraska, USA). The evaluations were performed in fully expanded leaves, on the central region of the 10th leaf in Stage 1, and in the closest leaf to the first ear in Stages 2 and 3 (10th or 11th leaf). The A, gS, E, and Ci/Ca measurements were performed between 9:00 and 11:00 am under constant photosynthetically active radiation (PAR, 1000 µmol photons m-2 s-1), atmospheric CO2 concentration (Ca) (400 ± 25 µmol mol-1), and at environmental temperature (25,8 ± 1 ºC) and relative humidity (74 ± 12 %). For RD measurement, the leaves were darkadapted for at least 5 h, and the evaluation was performed after 10:00 pm. The respiration were assessed the night before photosynthetic measurements.

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2.2.2 Chlorophyll a fluorescence Variables of chlorophyll fluorescence were measured in the same leaf of the

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photosynthesis at 5 DAA using a modulated portable fluorometer coupled to IRGA. For

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chlorophyll fluorescence, the first evaluations were conducted on dark-adapted leaves,

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so the reaction centers were fully opened (all oxidized primary acceptors) with minimum heat loss. Under this condition, it was possible to estimate the initial fluorescence (F0), maximum fluorescence (Fm), and potential quantum yield of photosystem II (PSII = [(F0-Fm)/Fm] [25]. After the light adaptation of leaves the

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chlorophyll fluorescence before saturation pulse (F) and the maximum fluorescence in light-adapted leaves (Fm’) were evaluated, the minimal fluorescence in light-adapted

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leaves could be calculated as F0’ = F0/[((Fm-F0/Fm)+(F0/Fm’)] [26] to estimate the effective quantum yield of PSII [YII = (Fm’-F)/Fm’] [25]. The YII was also used to estimate the apparent electron transport rate, ETR = YII.PAR.Aleaf.0,5 [27], where PAR

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is the photons flow (µmol m-2 s-1) on the leaves; Aleaf the amount corresponding to the

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fraction of incident light that is absorbed by the leaves; and 0.5 the excitation energy fraction directed to the PSII [28]. The non-photochemical quenching coefficient [NPQ =

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(Fm-Fm’)/Fm’] was calculated according to Bilger and Björkman [29].

2.3 Cytogenetic analysis

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The meiotic stages were evaluated in the S1 (V10) stage when the cell divisions occurred in the tassel. In the following seasons (S2 and S3), pollen grains had been formed, so it was not possible to observe the meiosis. Approximately 10 young buds per plant were collected, 24 h after P+E application, fixed in Carnoy solution, and stored at -20 ºC. Anthers were hydrolyzed in HCl (5 M) for 10 minutes at room temperature and stained with 2% acetic orcein before being squashed. The material was covered with cover glass and analyzed in binocular biological microscope (Leica, model DM500) with digital video camera (Leica, Model ICC 50).

2.4 Pollen grain viability Flower buds were collected at the R1 stage for all treatments, regardless of fungicide application season, in order to avoid errors related to different ages between the plants. For each plant, about 20 flower buds were collected, fixed in Carnoy

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solution, and stored at -20 ° C. The evaluation of the possible effects of P+E on pollen viability was estimated by counting the mature pollen for each plant. Slides were prepared by macerating anthers from two floral buds in acetic carmine 1%. A 4x

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objective of the biological microscope was used to count pollen grains. Stained pollen

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grains were considered viable, whereas empty or slightly stained were considered not

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viable. Two slides were prepared for each experimental unit, and 1000 pollen grains were counted.

In order to avoid that pollen grains from different treatments fertilized the stigmas, manual pollination was carried out after the opening of the anthers (R1). The

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2.5 Pollen grain germination

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pollination occurred when the stigmas reached approximately 5 cm in length.

In vitro grain pollen germination was evaluated in the R1 stage (after the opening of anthers), regardless of the fungicide application stage. The culture medium

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was prepared with 550 mM sucrose, 1.27 mM Ca(NO3)2, 1 mM KNO3, 200 mM H3BO3

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according to Gibbon et al. [30]. Approximately 4 mg of fresh collected pollen grains were homogenously spread on a labelled glass slide, in duplicate, containing 100 µL of

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culture medium. The glass slide were kept in a saturated atmosphere Petri dish and incubated at 25ºC for 2 h. After the germination, the glass slide was covered with a cover slip and analyzed using a binocular microscope. The pollen grains were

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considered germinated when the pollen tube exceeded the diameter of the own pollen grain. Two replicates were prepared, and 300 pollen grains were counted per plant.

2.6 Hundred-grain weight In order to determine the hundred-grain weight, the ears were harvested

manually when the grains have reached physiological maturity. The hundred-grain weight was assessed on an analytical balance.

2.7 Statistical analysis The obtained data were submitted to factorial analysis of variance, and, when necessary, the means were compared by Tukey test (p < 0.05) using the Analysis System Program Variance (SISVAR, version 5.4) [31].

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3 RESULTS

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The fungicide pyraclostrobin + epoxiconazole did not affect the photosynthetic

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rate (A), stomatal conductance (gS), ratio between internal and external CO2 (Ci/Ca),

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transpiration rate (E), or respiration (RD) (Table 1), regardless to which stage it was applied. A significant effect was observed only between the timing for the variables gS and Ci/Ca. There was also no significant interaction between the fungicide application and tested times, indicating that the observed difference was a unique behavior of

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phenological stages evaluated in this study.

There was no effect of the fungicide P+E in the potential quantum yield of PSII

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(Fv/Fm), electron transport rate (ETR), effective quantum yield of PSII (YII), and the non-photochemical quenching (NPQ) (Table 2). Effect was observed between the times for these variables. There was no significant interaction between the fungicide P+E and

Table 1

Table 2

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the timing (V10, V14 and R1).

It was also not possible to verify meiotic changes in plants treated with the

fungicide P + E (Figure 1). The meiotic stages anthers observed in both the control and in the treated plants showed normal pattern of cell division, shape, and size of meiocytes (Figure 1 a - f).

Figure 1

The viability of pollen grains was affected by the application of fungicide P+E, regardless of the application time (Table 3, Figure 2 a-b). On the other hand, pollen grain germination was not affected by the fungicide or the stage (V10, V14 and R1) (Figure 2 c-d).

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Table 3

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Figure 2

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There was no effect on the P+E in hundred-grain weight in maize plants (Table

P+E and control plants (data not shown).

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4), and no differences were found between the grain produced by plants treated with

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Table 4

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4 DISCUSSION

Application of pyraclostrobin + epoxiconazole fungicides at commercial doses neither compromised nor improved the photosynthetic metabolism of maize plants due

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to the maintenance of photochemical steps of photosynthesis. Furthermore, the NPQ,

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which is an indicator of non-photochemical thermal dissipation under stressful conditions [32], was not changed by fungicide application, suggesting the plants were

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not under stress. The changes observed at different stages are due only to the morphological plant development. The increase in gS did not represent water loss or increase their photosynthetic rate, despite expectations. It probably occurred once these

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plants were grown in a climate-controlled greenhouse with mild temperatures (~25 ºC), high relative humidity (above 60 %), and soil at field capacity. Negative effects of epoxiconazole in chloroplasts have been described in the

literature. The thylakoids integrity as well as the electrons transport and oxygen evolution were affected in Galium aparine L. plants exposed to 150 and 250 g a.i. ha-1 of P+E [13]. In the present study, at the recommended dose of epoxiconazole for maize (37.5 g a.i. ha-1), the thylakoid integrity has possibly been maintained since the ETR, and YII were not affected by the treatment. This indicates that the toxicity of this product to thylakoids is probably restricted to tests with higher dosages than the commercial dose and at exposure conditions different from what happens in the field, as testing immersed leaf discs in fungicidal solution provides better tissue contact and allows higher uptake and interference.

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The respiratory rates remained stable even in treated plants. This goes against the mode of action of one of the active ingredients: the pyraclostrobin. This fungicide belongs to the strobilurin chemical group, and its mode of action in fungi is complex III

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blockage in the respiratory electron transport chain [11]. As the complex III remains in

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all eukaryotes, at least a small reduction in respiration was expected [12]. The

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consistency of the respiratory rate in this study may be due to the activation of an alternative electron transport route after the fungicide application. Another possibility is that the changes might not have been detected by the measurement methods used. Some authors indicate that the reduction in respiratory rate caused by pyraclostrobin can be

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minimized by a transient increase on oxidation alternative route (AOX) [17, 33]. The AOX is insensitive to fungicides and receives electrons directly from the ubiquinone,

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leading to the oxidation of water in a way that electrons do not pass through the cytochrome bc1 (complex III) and oxidase cytochrome (Complex IV) [34]. Therefore, we suggested that the maize plants in the present study have AOX that is able to

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maintain the reduction of oxygen at the mitochondrial electron transport chain even

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under exposure to pyraclostrobin. It suggests that the commercial dosage of the fungicide does not cause phytotoxicity on maize plants of the DKB 390 PROII hybrid.

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Anomalies caused by phosphonate and phosphite [35, 36] fungicides were observed in meiosis in Lycopersicon esculentum and Allium cepa, respectively. However, in both studies, an overdose of the fungicide was applied once the

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commercial dose was applied four times, which does not occur in the field. In this study, considering the recommended dose of the fungicide, it was not possible to observe changes in meiosis of maize plants exposed to pyraclostrobin + epoxiconazole. Anomalies in meiosis may cause losses in the viability of pollen grains [16],

which was not observed in this study. This indicates that the fungicide did not affect the pollen grain formation during cell division. The inviability caused by pollen of plants treated with pyraclostrobin + epoxiconazole may have occurred due to the toxicity of the triazole to phytosterols, as noted in Gallium aparine L. plants [13]. According to the authors, the epoxiconazole causes a reduction in the campesterol and sitosterol concentration as well as changes in the ratio between them. Schaeffer et al. [15] found that low concentrations of sitosterol compared to campesterol may have been responsible for the reduction in the pollen grain viability, since Arabidopsis plants (in this case) produced less seeds and dead pollen grains. Thus, both the reduction in

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concentration as the change in the ratio of phytosterols are important factors in the pollen grain viability. The pollen viability was reduced by 1.4 % in the plants treated with P+E, which is probably due to a possible change in membrane structure caused by

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epoxiconazole.

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Considering that a vigorous maize plant can produce 30 to 60 million pollen

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grains [37], the viability reduction of 420 to 840,000 pollen grains could compromise productivity under field conditions. However, Uribelarrea et al. [38], evaluating different maize genotypes and under different plant densities, have found that even with a reduction of 50 % in pollen production, no changes in the kernel set were detected.

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Similar data were observed by other authors that did not attribute the decrease in grain production of maize to the reduction of pollen grain viability. Factors such as water

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deficit during the pollination period [39] or even the appearance of late receptive stigmas, which confers asynchrony between pollination and the ear formation [40], may be responsible for losses in production. Furthermore, in this study, although pollen

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viability was affected, no changes were observed in the pollen germination of maize

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plants.

Other fungicides from different chemical groups interfere with the viability or

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germination of pollen morphology in crops. Çali and Candam [41] evaluated the phosphonate effect in Solanum lycopersicum and observed that the fungicide caused a decrease in viability and alterations in pollen morphology. Also, the fungicidal effect of

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different chemical groups such as triazole, benzimidazole, and dinitrophenol on in vitro pollen germination was studied in Prunus persica and P. persica var. nucipersica [42] and Brassica campestris [43]. The authors reported that all fungicides negatively interfere with the pollen grain germination and concluded that pollen grains are very sensitive to toxic substances. However, in these works, the interference of the fungicide was tested directly in the culture medium without considering that when applied on the plant in the field, there are physical barriers against absorption such as the floral bud and anthers, which considerably reduces the direct interference of fungicide in the pollen grain. The hundred-grain weight is an important component of maize production, since it estimates the degree of grain filling [44]. The fungicide P + E did not affect the hundred-grain weight of maize plants of this study, which was expected, since the fungicide did not adversely affect the physiological status of the plants. We observed

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that the grain filling of the maize plants was not changed, indicating that in the absence of diseases, there is no increase in this parameter by applying P + E. Similar results were found by Ecco et al. [45] and Vilela et al. [46]. These authors evaluated other

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maize hybrids exposed to the fungicide pyraclostrobin + epoxiconazole with

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commercial doses and did not observe alterations in agronomic components; they even

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detected reduced incidence of foliar diseases. The authors found that, in low disease pressure, the fungicide did not increase any productivity parameter. Some studies suggest that strobilurin promotes physiological benefits and enhance yield by improving plant performance of maize [47], soybeans [48], and wheat

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[49]. Nevertheless, these authors do not always make it clear that the experiments were conducted using plants infected with fungi; therefore, it is expected that plants treated

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with fungicides show better performance when compared to the untreated infected control. Many of these authors even describe their studies as if they had worked in the absence of diseases, which was not observed in the severity indexes of their data. On the

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other hand, in other experiments with the same crops [46, 50, 51] without significant

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disease pressure, either physiological alterations or increased yield were reported. These authors describe that fungicide applications must be conditioned to disease emergence

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in order to ensure that there is an increment in production due to the treatment. Blandino et al. [6] concluded that the best strobilurin + triazole timing application in order to control diseases in maize considering only one application is at the flowering (R1) stage

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when the plant develops maximum photosynthetic activity. The fungicide promotes greater increases in yield when applied to plants with high disease severity or more sensitive hybrids [23], for acting on controlling the disease and enabling production when compared with the control under biotic stress. Considering the data obtained in this study, it is emphasized that the decision on the application of strobilurin + triazole in maize should be evaluated for disease pressure, so that the producer can obtain economic benefit by increased yield. Furthermore, there were no beneficial effects from fungicide application on the physiology of the maize plants grown without the actual incidence of diseases.

5 CONCLUSION

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The fungicide pyraclostrobin + epoxiconazole at commercial dose resulted in lower pollen grain viability, which did not change the physiology and production of the

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maize hybrid DKB 390 PROII.

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Acknowledgments

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The author thanks the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, grants no. 551456/2010-8 and 552689/2011-4) and the Instituto Federal Goiano - Campus Rio Verde for providing financial support. V.B. Junqueira, C. Müller and P.F.

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Superior (CAPES) for scholarships.

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Batista are grateful to the Coordenação de Aperfeiçoamento de Pessoal de Nível

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[45] M. Ecco, J.S. Rosset, L. Rampim, A.C.T.D. Costa, M.D.C Lana, J.R. Stangarlin, M.V.M. Sarto, Características agronômicas de híbridos de milho segunda safra submetidos à aplicação de fungicida, Agrarian 7 (2014) 504-510.

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wheat, Plant Pathol. 50 (2001) 190-205. [50] C. Swoboda, P. Pedersen, Effect of fungicide on soybean growth and yield. Agron.

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J. 101 (2009) 352-356.

[51] R. Weisz, C. Cowger, G. Ambrose, A. Gardner, Multiple mid-atlantic field experiments show no economic benefit to fungicide application when fungal disease is

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absent in winter wheat, Phytopathology 101 (2011) 323–333.

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Figure Captions

Figure 1. Meiotic phases of maize plants exposed to fungicide pyraclostrobin +

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epoxiconazole (a – e) and the untreated control (f) at growth stage V10. a) Metaphase I;

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b) Anaphase I; c) Metaphase II; d) Anaphase II, e) Telophase II and f) Anaphase II

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(control plants). Bar = 50 µm.

Figure 2. Pollen grain viability (a and b) and germination (c and d) in maize plants subjected to control (a and c) and fungicide pyraclostrobin + epoxiconazole (b and d)

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treatments. Blue arrows indicate viable pollen grains (a and b) or germinated (c and d); red arrows indicate inviable pollen grains (a and b) or not germinated (c and d). Bar = 2

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µm.

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Table 1. Net photosynthetic rate (A), stomatal conductance (gs), transpiration rate (E), the ratio between internal and external CO2 concentration (Ci/Ca), and the dark respiration (RD) of maize plants exposed to pyraclostrobin + epoxiconazole (P+E)

3

s-1

s-1

Control

26.70±1.29

0.14±0.02b

P+E

26.58±2.56

0.18±0.03b

Control

27.58±2.52

0.22±0.04a

P+E

26.95±2.17

0.20±0.03a

Control

27.75±0.76

P+E

27.17±3.23 n.s.

Stage

n.s.

X

Gourp Stage

n.s.

Block

n.s.

C.V. (%)

8.65

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Ci/Ca

RD

mmol H2O m-

μmol CO2 m-

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mol H2O m-2

E

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μmol CO2 m-2

Group

2

s-1

2

s-1

0.29±0.08b

2.09±0.31

0.69±0.19

0.32±0.14b

2.23±0.36

0.63±0.13

0.42±0.08a

2.19±0.32

0.64±0.10

0.39±0.07a

2.05±0.17

0.69±0.16

0.19±0.02ab

0.34±0.06ab

2.32±0.20

0.65±0.11

0.22±0.05ab

0.43±0.10ab

2.63±0.52

0.80±0.14

n.s.

n.s.

n.s.

n.s.

*

*

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

16.97

20.93

15.52

20.46

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gS

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A

D

Stage

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fungicide at three growth stages, five days after the treatment application.

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* Significant by factorial analysis (p ≤ 0.05). Mean ± SD (n=5) followed by the same letter, in the column, do not differ significantly from each other as

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determined by Tukey’s test (p ≥ 0.05)

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Table 2. Potential quantum yield of PSII (Fv/Fm), electron transport rate (ETR), effective quantum yield of PSII (YII), and non-photochemical quenching coefficient (NPQ) of maize plants exposed to pyraclostrobin + epoxiconazole (P+E) fungicide at

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1.20±0.13a

0.37±0.02a

1.21±0.09a

143.35±8.79b

0.34±0.02b

1.05±0.11b

0.78±0.01b

142.33±6.28b

0.34±0.01b

0.95±0.19b

Control

0.78±0.00b

145.63±7.42b

0.35±0.02b

1.11±0.20ab

P+E

0.78±0.00b

147.05±10.18b

0.35±0.02b

1.20±0.18ab

n.s.

n.s.

n.s.

**

**

*

0.79±0.00a

157.07±6.20a

P+E

0.79±0.00a

156.18±8.58a

Control

0.78±0.01b

P+E

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Control

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0.37±0.01a

ETR

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NPQ

Fv/Fm

Group

n.s.

Stage

**

Gourp X Stage

n.s.

n.s.

n.s.

n.s.

Block

n.s.

n.s.

n.s.

n.s.

C.V. (%)

5.63

5.68

14.48

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1

YII

Group

D

Stage

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three growth stages, five days after the treatment application.

0.47

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**, * Significant by factorial analysis, p ≤ 0.01 and p ≤ 0.05, respectively. Mean ± SD (n=5) followed by the same letter, in the column, do not differ significantly from each other as

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determined by Tu ey’s test (p ≥ 0.05)

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Table 3. Pollen grain viability and germination of maize plants exposed to pyraclostrobin + epoxiconazole (P+E) fungicide at three growth stages. Pollen grain viability

Pollen grain germination

Control

96.75±1.19a

63.13±4.83

P+E

95.20±1.07b

Control

96.88±1.77a

P+E

95.54±1.54b

Control

97.68±0.63a

61.59±5.80

P+E

96.22±1.03b

63.48±4.92

Group

**

n.s.

Stage

n.s.

n.s.

X

Gourp Stage

n.s.

n.s.

Block

n.s.

n.s.

1.29

7.66

3

C.V.(%)

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66.88±4.91

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2

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1

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Group

MA

Stage

65.03±1.73 69.20±5.54

** Significant by factorial analysis (p ≤ 0.01).

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Mean ± SD (n=5) followed by the same letter, in the column, do not differ significantly from each other

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as determined by Tu ey’s test (p ≥ 0.05)

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Control

41.89±4.75

P+E

40.84±1.49

Control

43.57±2.25

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Group

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Stage

2 P+E

44.38±3.49

Control

Group

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Stage

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3 P+E

41.78±1.82 42.42±4.27 n.s. n.s. n.s.

Block

n.s.

C.V.(%)

7.67

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Group X Stage

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1

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Figure 1

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Figure 2

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Graphical abstract

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HIGHLIGHTS

The fungicide application did not affect the photosynthetic efficiency on maize disease-free plants.



The reduction in pollen grain viability did not affect maize production.



The physiological, reproductive development, and production effects were similar regardless of the plant stage in which the fungicide was applied.

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