Potential of the Dithiocarbimate Fungicides on the Control of Coffee Leaf Rust and Asian Soybean Rust

Coffee leaf rust (Hemileia vastatrix) and Asian soybean rust (Phakopsora pachyrhizi) are diseases that cause great losses in the productivity of these crops, not only in Brazil but on a global scale. Coffee and soybean varieties grown are susceptible to these diseases. Thus, it is necessary to search for efficient compounds for their chemical control, mainly from the group of protectors or residuals so that they can be formulated with systemic fungicides to control the diseases. This allows not only the efficient management of diseases but also the prevention of the emergence of resistant mutants in the populations of these pathogens. In this context, the present study aimed to evaluate the sensitivity and effect of bis(N-R-sulfonyldithiocarbimato)zincate(II) salts fungicides on the epidemiological components of pathosystems coffee × H. vastatrix and soybean × P. pachyrhizi. Initially, four zinc(II) complexes salts (1A, 2A, 1B, and 2B) with N-R-sulfonyldithiocarbimates were synthesized. In the first experiment, the in vitro sensitivity of H. vastatrix and P. pachyrhizi was studied for the four compounds synthesized and mancozeb at 0.5, 5.0, 50.0, 100.0 and 200.0 μmol L. All the compounds synthetized in this study had inhibitory effects on H. vastatrix and P. pachyrhizi. In the greenhouse it was studied the effect of bis(N-R-sulfonyldithiocarbimato)zincate(II) salts on the epidemiological components of coffee leaf rust and Asian soybean rust. For the pathosystem coffee × H. vastatrix, there were no differences in the values obtained for the bis(N-R-sulfonyldithiocarbimato)zincate(II) salts and mancozeb for the latent period. For the sporulated lesion variable, the control treatment had a mean value of 149.0 lesions/leaf, differing significantly from the other treatments. The mean value of compound 2B was estimated as 25.0 lesions/leaf, differing significantly from treatments 1A, 1B, 2B, and mancozeb. Treatments 1A, 1B, 2B, and mancozeb did not differ significantly from each other. For the Asian soybean rust, the area under the disease progress curve had a mean value of 75.8 for the control, while for the 2A treatment the value was 4.1, differing from the other compounds. The treatments 1A, 1B, 2A, and mancozeb did not differ significantly from each other. In conclusion, compounds 1A, 2A, and 1B were more efficient in the control of the coffee leaf rust, while compound 2A was efficient in the control of the Asian soybean rust.


Introduction
The increase in the world population has driven the search for new agrochemicals to ensure the sustainable production of food. Such agrochemicals aim to maintain the productivity potential of crops, not only in Brazil but on a global scale. In Brazilian agribusiness, the production of coffee (Coffea arabica L.) and soybean (Glycine max L. Merryl) correspond 45.3 milions bags and 120.3 milions tons, respectively (CONAB, 2019). Among the main factors capable of limiting coffee and soybean production in the Brazil and world, rust diseases stand out. The fungus Hemileia vastatrix Berk. & Br., the causal agent of coffee leaf rust, causes damage that can affect up to 50% of coffee production (Zambolim, 2016). Besides, the fungus Phakopsora pachyrhizi, the causal agent of Asian soybean rust, causes damage of up to 90% to soybean crops (Yorinori et al., 2005). Management strategies for these diseases are based on a combination of specific-site and multi-site fungicides (Godoy et al., 2016;Zambolim, 2016;Reis et al., 2021). Usually, for the coffee leaf rust, strategies are based on the use of triazole fungicides formulated with strobilurins combined with multi-site copper fungicids. However, copper is a heavy metal that can accumulate in the environment, despite being crucial in the control not only of coffee leaf rust but also of other diseases and as a nutrient (Sousa et al., 2017). For the Asian soybean rust, chemical control strategies usually adopt triazoles, strobilurins, and dicarboxamides associated with multi-site chlorothalonil or mancozeb (Ponce et al., 2019). The use of multi-site fungicides has become indispensable to reduce the selection pressure on the pathogen population. Several studies of multiple resistance to triazole, strobilurin, and dicarboxamide groups are reported in the literature (Schmitz et al., 2014;Twizeyimana & Hartman, 2017;Simões et al., 2018). Currently, the companies have been using multi-site fungicides developed in the 1950-60s (Zambolim & Caixeta, 2018;Reis et al., 2021).
Several studies have shown, in vitro, the inhibition of mycelial growth of the fungi Colletotrichum gloeosporioides and Botrytis cinerea by new compounds belonging to the dithiocarbimate group (Bottega et al., 2013;Oliveira et al., 2015). The dithiocarbimate group is analogous to the dithiocarbamates but does not have a commercial formulation. The dithiocarbamates are widely used in agriculture, and their leading representative is mancozeb (Zambolim, 2008). Such groups differ chemically only by the existence of a double bond between the carbon and nitrogen atoms. In this context, researches seeking for new fungicide compounds that are highly efficient in disease control and have good acceptance by the society are vital. Dithiocarbimates are promising compounds since they have a multi-site action, either alone or as compound formulations with systemic fungicides, in the disease control of plants. The first study, involving this group of chemical compounds, in the disease control was conducted by Vidigal et al. (2019). The authors reported for the first time the effect of new heteroleptic nickel(II) complexes with aromatic and aliphatic sulfonyldithiocarbimates, triphenylphosphine and 1,2-bis(diphenylphosphine)ethane on the uredospore germination of H. vastatrix and P. pachyrhizi; however, their experiments were restricted to in vitro assays, and the control efficiency of this fungicide group in controlled conditions in vivo is unknown.
This work is justified due to the fact that, coffee leaf rust and Asian soybean rust can severely damage both crops. In addition, there is a great need to find new protective fungicide compounds to be used in mixtures with systemic and mesostemic fungicides, aiming to reduce the emergence of resistant mutants of specific mechanism of action fungicides, used in disease control.
Thus, this study aimed to evaluate the sensitivity and effect of bis(N-R-sulfonyldithiocarbimato)zincate(II) salts on the epidemiological components of the pathosystems coffee × H. vastatrix and soybean × P. pachyrhizi.

Synthesis of N-R-Sulfonyldithiocarbimate Potassium Salts
The synthesis of the four N-R-sulfonyldithiocarbimate potassium salts consisted of two stages, following the procedures described by Amin (2011).
The first stage included the production of the precursors of potassium dithiocarbimates. In a round-bottom flask, 10,000 µmol of methanesulfonamide or 4-chlorobenzenesulfonamide; 20 mL of N,N-dimethylformamide; 0.60 mL of carbon disulfide (10,000 µmol) and 1.32 g of KOH (20,000 µmol) were added. The reaction mixture was kept under stirring until the consumption of KOH, indicating the end of the reaction. Then, 20 mL of absolute ethanol was added, and the round-bottom flask was placed in an ice bath.
The yellow precipitates resulting from the reaction were filtered and then washed with ethanol, ethyl acetate, and diethyl ether. Soon after, the precipitates were left in a desiccator for 24 hours. At the end of this reaction, the potassium methylsulfonyldithiocarbimate and the potassium 4-chlorophenylsulfonyldithiocarbimate were obtained as products.
The second stage consisted of the stabilization of the dithiocarbimates from the formation of zinc complexes. To perform the synthesis of the zinc complexes, 1,000 µmol of potassium dithiocarbimate, 20 mL of MeOH/H 2 O solution (1:1 v/v), and 500 µmol of zinc sulfate dissolved in 4 mL of H 2 O were added in a round-bottom flask. The reaction mixture remained under stirring for one hour. Then 1,000 µmol of tetraphenylphosphonium chloride or tetrabutylammonium bromide dissolved in water were added, drop by drop. The resulting precipitate was filtered in a funnel with a porous porcelain plate, washed five times, and then dried under reduced pressure for 24 hours. In this way, "1" is 4-chlorophenyl group and "2" is methyl group, white group "B" has the cation tetrabutylammonium and group "A" has the cation tetraphenylphosphonium. The four synthesized compounds were named as follows: tetraphenylphosphonium bis (

Production of H. vastatrix and P. pachyrhizi Uredospores
Coffee seeds of the variety Caturra lineage IAC 144 were sown in the sand and kept at 28 °C. After thirty days, the seedlings were transplanted to 2.0 dm 3 vessels and cultivated for 45 days. Then, the third pair of leaves were inoculated with the H. vastatrix uredospores race II from the Plant Protection Laboratory, Department of Plant Pathology, Federal University of Viçosa, state of Minas Gerais, Brazil. The plants remained for 48 hours in a dew chamber in the dark at 22 °C during the entire experiment and were kept in a growth chamber with a 12-hour photoperiod for 35 days until the production of uredospores.
The uredospores of P. pachyrhizi from the Plant Protection Laboratory were inoculated at the V5 phenological stage in the soybean variety TMG 132. Subsequently, the plants in 2.0 cm 3 vessels were kept for 48 hours in the dew chamber, in the dark, and at 22 °C. After inoculation, the plants were placed in a growth chamber with a 12-hour photoperiod for 15 days to produce uredospores.
The produced uredospores were collected. One part was kept in a desiccator at 5 °C and 50% relative humidity and the other part in an ultra-freezer at -80 °C.
The compounds 1A, 2A, 1B, and 2B (0.01 g) were dissolved into 10 mL of dichloromethane, and the volume corresponding to each concentration was transferred to 50 mL round-bottom flasks. The solvent was evaporated under reduced pressure. DMSO and Tween 80 were added to each round-bottom flask at 0.5% (v/v).
These solutions were homogenized in 2% water agar, and the mixtures were distributed in 60 × 15 mm Petri dishes. A 0.1 mL aliquot of 10 5 uredospores mL -1 from H. vastatrix or P. pachyrhizi was spread on each plate with a Drigalski spatula. The plates were kept at 25 °C under continuous darkness in a BOD Incubator for 24 hours. After this period, the germination of 100 uredospores per plate was randomly evaluated by optical microscopy at 400-fold magnification. The mean values of the in vitro sensitivity experiment were used to calculate the inhibition frequency of uredospore germination by employing the equation: where, G c = Spores germinated in control treatment with DMSO and Tween 80; G i = Total germination of each observation.

Effect of bis(N-R-Sulfonyldithiocarbimato)Zincate(II) Salts on the Epidemiological Components of the Coffee Leaf Rust and Asian Soybean Rust
For the tests of coffee leaf rust, coffee seedlings of the Caturra variety (susceptible) at four months of age for the Asian soybean rust, soybean seedlings at 40 days of age were used.
Two in vivo experiments were performed in a completely randomized design with three replicates, each including three 3.0 dm 3 vessels with three plants. The treatments consisted of compounds 1A, 2A, 1B, 2B and the fungicide mancozeb. To evaluate the effect of the compounds on H. vastatrix (coffee leaf rust), the mancozeb was used at 1,000 µmol L -1 ; for P. pachyrhizi (Asian soybean rust), 50 µmol L -1 of water and Haiten adjuvant (Arysta ®) at 0.1 mL.L -1 were used as two negative controls. Therefore, in both experiments, seven treatments and three replicates were conducted.
In the experiment with coffee leaf rust, chemical compounds were applied to the abaxial face of coffee plants, on the third and fourth pairs of leaves, using a 100 mL manual atomizer. After 24 hours, the plants were inoculated by sprinkling, using a DeVilbiss No. 15, with a suspension at 10 5 uredospores mL -1 . They were kept for 48 hours in the dark, in a humid chamber at 22 °C. After this period, the plants were incubated in a chamber at 22 °C, with a 12-hour photoperiod. Evaluations were performed every two days, at the 18 th day after inoculation. The evaluated response variables were as follows: incubation period (IP); latent period (LP); spore production (SP); number of lesions sporulation (NLS); area under the disease progress curve (AUDPC).
In the experiment with the Asian soybean rust, chemical compounds were applied in the third pair of soybean plants, using a 100 mL manual atomizer. After 24 hours, the plants were inoculated by sprinkling, using a DeVilbiss No. 15, with a suspension at 10 5 uredospores mL -1 . They were kept for 48 hours in the dark, in a jas.ccsenet.
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The     For P. pachyrhizi, the lowest IC 90 value in experiment 1 was 6.0 µmol L -1 for compound 1B. In experiment 2, the lowest value was 6.0 µmol L -1 for mancozeb, followed by 8.0 µmol L -1 for the compound 1B ( Table 2). The lowest doses of mancozeb, 1A, and 1B inhibited more than 50% of uredospore germination (Table 2; Figure 3). The analysis of the IC 90comb of the compounds showed no significant differences between the treatments 1A, 1B, 2B, and mancozeb (standard control) ( Table 2). The lowest performance was observed for the compounds 2A, which differed significantly from other treatments.   Figure 4 shows the result of the epidemiological components when Caturra coffee plants were inoculated with H. vastatrix uredospores. The lowest PI for the control with water was 16 days; the highest values were achieved for the compounds mancozeb, 2A, and 2B, which lead to expressed symptoms at 24 days. All treatments differed from the control with water. The PI of the compounds did not differ significantly from the mancozeb (standard control) (Figure 4).     (Leclerc et al., 2014).

Effect of Zinc(II) Complexes Salts on the Epidemiological Components of H. vastatrix
The higher estimated values of the epidemiological components IP and LP concomitantly with lower SP value found in this study suggest that the use of that bis(N-R-sulfonyldithiocarbimato)zincate(II) salts may reduce the progress rate (r) of the coffee leaf rust epidemic, and most likely have a lower impact on coffee productivity (Zambolim, 2016). On the other hand, the 2B compound had lower estimated LP values combined with higher SP values for coffee leaf rust. Thus, the compound 2B should be disregarded in new studies since the epidemiological components indicate risks of inefficiency in the control of coffee leaf rust. When chemical control is inefficient, the disease causes defoliation in coffee plants and consequently reduces the yields of crops in the following year (Souza et al., 2011;Talhinhas et al., 2017).
The results of the variables LP, pustules/cm 2 , RCE, and AUDPC showed that treatment with compound 1A was not efficient to control the Asian soybean rust. On the other hand compound 2A and mancozeb were efficient and did not differ significantly from each other. However, the efficiency of compound 1A may increase at higher doses. In the present study, the bis(N-R-sulfonyldithiocarbimato)zincate(II) salts were used in the molar ratio to standardize the number of compounds in each treatment. Thus, the molar concentration of each compound used in the experiment (50 µmol L -1 ) was equivalent to a reduction of 175 times concerning the recommendation of 3.75 kg of the active ingredient mancozeb per hectare.
Compounds 1B and 2B caused phytotoxicity in soybean leaflets. However, there were no visible anomalies in the leaf tissues submitted to treatment with compounds 1A and 2A. The phytotoxicity property of the compounds group B may be related to the presence of different counter-ions. Group A contains tetraphenylphosphonium, and group B contains tetrabutylammonium. Besides, phytotoxicity may be related to the interaction of ammonium ion in leaf tissues. The plant cells absorb the ammonium ion passively; however, if the absorption levels are higher than those of metabolization, the intoxication of the leaf tissues may occur (Barker, 1999). Usually, damage caused by the ammonium ion affects photosynthetic processes, ATP synthesis, and the electron transport chain (Opanasenko & Vasyukhina, 2009).

Conclusion
The salts "bis(N-R-sulfonyldithiocarbimato)zincate(II)" are an option for the control of coffee leaf rust and Asian soybean rust; their efficiency is comparable to the standard product mancozeb. For Asian soybean rust, only the compound 2A was efficient for the control. Compounds 1A, 2A, and 1B were adequate for the control of coffee leaf rust.