Addition of Mancozeb to DMI + QoI, and SDHI + QoI Co-formulations Improving Control of Asian Soybean Rust

The reduced sensitivity of Phakopsora pachyrhizi to site-specific fungicides used to control Asian soybean rust by the current co-formulations needs investigation. To improve the rust control the performance of cyproconazole + picoxystrobin, tebuconazole + picoxystrobin, cyproconazole + azoxystrobin, epoxyconazole + pyraclostrobin, fluxapyroxade + pyraclostrobin, benzovindiflupyr + azoxystrobin, prothioconazole + trifloxystrobin and cyproconazole + trifloxystrobin mixtures added by five doses of the multisite mancozeb were evaluated. The fungicides were sprayed at four growth stages the first performed at R1 growth stage and the others with 15-18 days intervals. The rust severity was quantified, the control was calculated, the percentage of chlorophyll and the yield of soybean were determined. The mean of rust control by the mixtures without addition of the multi-site fungicide was 46% (21 to 71%). There was an increase in control efficiency due to addition and mancozeb doses in all treatments. Control over 80% was obtained with tebuconazole + picoxystrobin, fluxapyroxade + pyraclostrobin, benzovindiflupyr + azoxystrobin, and prothioconazole + trifloxystrobin added at least of 2.0 kg/ha mancozeb. In unsprayed plots the maximum 78% severity corresponded to 59% damage. There was an increase in chlorophyll content and soybean yield as a function of the mancozebe increased doses: 2,019 kg/ha in the unsprayed control and in the best treatment 5,132 kg/ha. Actual control reduction due to fungal decrease in sensitivity can be improved by the multi-site fungicide addition.

In Brazil, ASR chemical control started in the 2002/03 season. The first applied chemicals were triazoles or demethylation inhibitors (DMI) mainly flutriafol and tebuconazole solo. Difenoconazole, myclobutanil and tetraconazole with the same single-site mode of action were also used in a lesser extent. Later, cyproconazole, epoxiconazole and tebuconazole were used only in mixtures with QoIs azoxystrobin, pyraclostrobin, and trifloxystrobin (Godoy & Palaver, 2010). control higher than 80%, thus, with 37% mean control losses due to control failure can threaten soybean economical sustainability (Godoy & Palaver, 2011;Godoy et al., 2015;Reis et al., 2015Reis et al., , 2017. ASR control reduction has been reported in Brazil and fungus sensitivity reduction towards DMI, QoI and their mixtures has been shown (Fundação, 2008;Silva et al., 2008;Blum, 2009;Godoy et al., 2015). Mutation Cyp 51 towards DMI, and F129L to QoI have been reported (Schmitz et al., 2014;Klosowski et al., 2016), and on August 7 th , 2017 the international Fungicide Resistance Action Committee (FRAC) warned on the presence of I86F mutation towards SDHI fungicides in sub unit C of SDH enzyme (Simões et al.,2018).
The threat that concerns soybean producers is the evolution season-after-season of the P. pachyrhizi reduced sensitivity to site-specific fungicides (Reis et al., 2017). The effectiveness of fungicide control of ASR and which results in maximum profit for the producer is over 80%, but currently it is less than 50% (Reis et al., 2017). When control is reduced by the development of resistance producers profits are also restricted (Main, 1977).
We hypothesized that the control failure and the evolution of control reduction, season after season is due to the cross and multiple resistance of P. pachyrhizi towards DMIs, QoIs and SDHIs fungicides can be partially reversed by the addition of multi-site fungicide.
The objective of this work was to compare the performance of commercial site-specific co-formulations with and without the addition of mancozeb a multisite mode of action fungicide in an attempt to recover ASR control efficacy.
Sprayings were performed with a backpack sprayer pressurized by CO 2 delivering 150 L/ha. Boom, with four Hypro DB015F120 nozzles 0.5 m apart was kept 40 cm above plant canopy. Four sprayings were programmed the first at growth stage (GS) R1 with zero leaflet incidence and the other with 15 to 18 days interval.
Experimental design was a factorial with four mancozeb rates × eight co-formulations (DMI + QoI and QoI + SDHI) in 2.25 × 6.0 m plots, in a randomized design with four repetitions. Four extra treatments with 0.0, 1.0, 1.5, 2.0, 2.5 and 3.0 kg mancozeb/ha were added where 0.0 manzozeb as the unsprayed treatment.
Two different methods for disease assessment were performed. To detect ASR onset the leaflet incidence was used as the most sensitive method. For severity central leaflet of leaves inserted in the main stem of 10 random plants per plot were taken weekly and analyzed under a stereomicroscope. Leaflet intensity was appraised according to Godoy et al. (2006) severity scale.
The relative chlorophyll content was measured with a chlorophyll meter (SPAD-502, Minolta, and Osaka, Japan) (Minolta, 1989) which measures the percentage of chlorophyll in the leaf blade. The readings were performed in five leaflets per plot of the upper leaflets and taking four readings per leaflet.
Plots were mechanical harvested with a Wintersteiger plot combine in 13.5 m 2 /plot. Data were submitted to analysis of variance and the treatments means compared by Tukey's test (p < 0.05) for severity and for grain yield. Regression analysis between grain yield and mancozebe rate was also performed. The extra treatments were not included in the complete statistical factorial analysis.

Results and Discussion
To detect the ASR onset and plots progress curve the disease was measured by leaflet incidence a sensitive method. First spraying was performed on January 12 th , 2016 at R1 GS. Rust was first found on January 28 th with 49% leaflet incidence coincided with the time of the second spraying, 16 days after rust onset. In 41 days, rust epidemics increased from zero to 100% leaflet incidence ( Figure 1). jas.ccsenet.
The highes (1.5, 2.0, 2 The lowes 1.5 kg/ha mancozeb- In 2018/19 season control for prothioconazole + trifloxistrobin was 41%. Control of benzo + azoxi (63%) shows reduction in relation to previous season. The overall rust control mean by the eight most used mixtures solo due to P. pachyrhizi sensitivity reduction to the DMIs, QoIs, and SDHIs, was 46% (21 to 71%) ( Table 2) considered very low. It should be reinforced that > 80% control is required to equalize the costs of fungicide application (Boller, 2010).
Chlorophyll content in unsprayed treatment was 15% and for mancozeb solo rates in extra treatments were 17, 18, 20, and 20. Considering the chlorophyll content the interaction between co-formulations alone and mancozebe doses was significant (p = 0.05). The overall chlorophyll content for the co-formulations alone was 27% while for the addition of 3.0 kg/ha mancozeb increased to up 33% (23 to 42%). Considering the minimum and the maximum content there was an increase from 19% (epox + pyra) up to 42% (prot + trif + mancozeb). The chlorophyll content can also be increased by strobilurin effect on soybean plant physiology (Fagan et al., 2015) as well for manganese nutritional effect as shown in wheat plants (Reis & Floss, 1980). It is likely that the effect on turning the plants greener can have a reflection on the grain yield. Note. Chlorophyll content (%) in the control treatment-15%.
After six seasons of the DMIs use alone in the control of soybean rust, growers complained on the control failure. At that moment, Silva et al. (2008) reported the control reduction in experiments, and later was confirmed by Reis and Deuner (Reis et al., 2015) in the P. pachyrhizi sensitivity reduction measured by the IC 50 . Later, Xavier et al. (2015) demonstrated the presence of cross-resistance among DMIs.
Regression analysis between grain yield and mancozebe rates showed y = 220.09x + 3,798.3, R 2 = 0.83 [where 'y' is soybean grain yield (kg/ha), and 'x' = kg/ha mancozebe, R 2 coefficient of determination] showing that each 0.5 kg/ha mancozeb increment increased 220.09 kg/ha soybean grain yield. In our experiment the damage (sensu Nutter et al., 1993) in the soybean yield was 59% considering the productivity in the unsprayed plots (rust severity 78%) of 2,019 kg/ha and 4,920 kg/ha the highest overall average (21% control).
On March 8 th , 2017, FRAC released in internet a note on the P. pachyrhizi resistance to carboxamides and that reduction in sensitivity could be crossed to members of this group. In our study, the control resulting from the application of benzovindiflupyr + azoxystrobin was only 63% showing the sensitivity of P. pachyrhizi to SDHI since it was previously 82% . This co-formulation was released in the market in 2014 growing season, therefore, being exposed to fungal directional selection for just three seasons. Therefore, SDHIs can be considered a high-risk chemical group and P. pachyrhizi a high risk fungus for resistance development.
Overall mean for rust control by the eight most used mixtures due to P. pachyrhizi sensitivity reduction to the DMIs, QoIs and SDHIs, was just 46%. It is accepted that for economic control 80% efficiency is required (Boller, 2010).
It should be noted that there is evidence that mancozeb has no potential to recover control of mixtures that have less than 50% efficacy when sprayed solo (Table 3). There is evidence that some co-formulation reaching < 52% control will no longer be effective, even by adding 3.0 kg / ha of mancozeb (Tables 3 and 4).
In view of the widespread reduction of soybean rust control by DMI + QoI, SDHI + QoI the mixtures it is suggested to use only ready mixtures containing DMI + QoI + multi-site, or SDHI + QoI + multi-site to reduce/paralyze directional selection.
Phakopsora pachyrhizi cross resistance towards DMIs, QoIs, and SDHIs alone and multiple resistance reach all of them solo or in mixtures (Xavier et al., 2015). Therefore, the triple co-formulations (DMI + QoIs + SDHIs), now most used in Brazil, would show a better performance in the rust control than their isolated components?
The use of multi-site fungicides to fight resistance is not a new strategy (Gullino et al., 2010;FRAC, 2015FRAC, , 2020. It has been used with success in the control of potato and tomato late blight and of downy mildew in grapes (FRAC, 2020;Gulino et al., 2010;Muchiri et al., 2009). Our results confirm the potential of mancozeb to improve ASR control as it was performed with Phytophthora infestans (Mont.) de Bary. Multisite fungicides remain essential for management of fungicide resistance in and Plasmopara viticola (Berk. & Curtis) Berl. & De Tonia (Corio-Costet, 2012).
Our results are promising to improve ASR control but how many soybean seasons will be needed to growers be aware and treat the whole area (39.6 million ha) with the site-specific double or triple co-formulations added by a multi-site fungicide to reduce directional selection?
Finally, to reduce directional selection the whole soybean grown area and all sprayings should be performed with co-formulations containing site-specific + multi-site mix fungicide (chlorothalonil, mancozeb, copper oxychloride).