In the State of Sinaloa, Mexico, white mold is a disease affecting bean plants in any stage of development, its incidence and severity varies with the winter moisture and fungus populations in the soil, reduce performance reaches up to 75%, hence it is considered the most important fungal disease of this crop (^{Apodaca et al., 2011}). Farmers try to manage the disease commonly by conventional fungicidal sprays, whose efficiency is often low because: a) the right ingredients are used; b) are applied with a bad coverage of the plants; c) and there may be selection for resistance to these pesticides, among other reasons. Therefore, the objective of this study was to determine the effectiveness of conventional fungicides and bio-rationales vs. in vitro S. sclerotiorum.

The study was conducted at the Laboratory of Plant Pathology, College of Agriculture UAS strongly Valley, Juan Jose Rios, Sinaloa, in the spring of 2013.

Isolation of S. sclerotiorum. Bean plants infected with white mold were collected, Valle del Fuerte, Sinaloa. To isolate the fungus-infected tissue pieces on the edge of the lesion at the cut stems and pods. The material was disinfected with sodium hypochlorite 5% for thirty seconds; then washed three times with sterile distilled water and dried with sterile paper towels. Finally five pieces of tissue from each sample were placed in a Petri dish with culture medium potato dextrose agar (PDA, Bioxon^®) and incubated at 18-20 °C. In order to obtain pure cultures, 24-48 h, the edge of the actively growing colonies, small portions of hyphal tips were cut using a sterile needle, which are being transferred to boxes with PDA. The purified isolates were preserved in culture tubes with PDA at 4-6 °C until use in bioassays.

Test with conventional fungicides. 10 fungicides (Table 1), which are recognized by SENASICA-SAGARPA control were evaluated: S. sclerotiorum in bean [com benomyl, boscalid + pyraclostrobin, carbendazim, fluazinam, thiophanate -methyl and pyrimethanil, among others (^{Apodaca et al., 2011})] and other commercial fungicides used against fungi from the soil or foliage in various plant species. Each fungicide was evaluated a1 least two concentrations (ppm); boscalid, pyraclostrobin and boscalid + cyprodinil + fludioxonil were evaluated at three concentrations, generating a total of 23 chemical treatments plus control water.

Table 1 Effect of conventional synthetic fungicides on mycelial growth of in vitro S. sclerotiorum. 

1. PHC Mil Stop, Plant Health Care; 2. Cantus, Basf; 3. Cabrio, Basf; 4. Bavistin, Basf; 5. Shogun, Syngenta; 6. Switch, Syngenta; 7. Vigold, Bayer; 8. Kasumin, Arysta; 9. Cueva, Mitsui de Mexico; 10. Sportak, Bayer. 11. Testigo agua. Los datos transformados a raíz cuadrada (√x+0.5) antes de análisis. Medias con misma letra en cada columna no son significativamente diferentes (Tukey α= 0.05).

Randomly selecting an isolate of S. sclerotiorum, same as aseptically transferred, the tube with PDA where it was preserved, the centre of plates with PDA. The dishes were incubated at a18-20 °C for five days. The fungicides were added to the desired concentrations in sterile flasks with fresh PDA when it was approximately 45 °C and immediately dispensed into disposable Petri boxes (8 cm diameter) sterilized. Immediately in the centre of each of the treated cases, a circular diameter of 1 cm-PDA was placed slice fungus. The control was established in PDA without fungicide.

Completely randomized design was used; the experimental unit consisted of a Petri dish seeded with fungus. The effect of treatments was assessed at 48, 96 and 144 h, measuring the radius of the fungus colony on the five replicates of each treatment.

Bio-rational fungicides trial. 10 ingredients (Table 2) were tested for their toxicity was unknown to S. sclerotiorum. The trial was conducted similar to that described for the synthetic fungicides way, but in this case was incubated at 14-16 °C; three replicates per treatment, and the effect of treatments was assessed at 48, 96 and 240 h.

Table 2 Effect of bio-rational fungicides on mycelial growth of S. sclerotiorum in vitro. 

1. Compuesto químico; 2. Serenade; 3. Oxidate; 4, 5, 6 y 7. Extractos artesanales. 8. Fractal; 9. Spectra10. Citripower; 11. Testigo agua. CAN= complejo de antibióticos naturales. Los datos se transformaron a raíz cuadrada (√x+0.5) antes de su análisis. Medias con la misma letra en cada columna no son significativamente diferentes (Tukey α= 0.05).

Data obtained from within the colony, were transformed to square root to homogenize the variances (^{Little and Hills, 1989}); then subjected to an ANOVA and arithmetic means were compared by Tukey test (p = 0.05) through the ^{SAS software, version 9.0 (2002)}.

Biological effectiveness of conventional fungicides. After 48 h of seeding the fungus, the smaller radius of the colony (p< 0.0001) of S. sclerotiorum was recorded in the concentrations evaluated boscalid (except 0.25 ppm) + pyraclostrobin, carbendazim, fluazinam, fludioxonil + cyprodinil, fluoxastrobin and prochloraz. In contrast, treatment with kasugamycin, copper octanoate and potassium bicarbonate (low dose), were similar to the control (Table 1).

At 96 and 144 hds boscalid + pyraclostrobin, carbendazim, fluazinam, cyprodinil + fludioxonil and prochloraz, at all concentrations tested, maintained the greater suppression of the colony (p <0.0001), as was observed at 48 hds. Potassium bicarbonate, 4.25 ppm, and the two dosages of kasugamycin and copper octanoate, allowed a similar or higher than the control development. The fluoxastrobin, at both concentrations tested, and boscalid (5. 0 and 0.5 ppm), with good control effect at 48 hds, allowed significant growth of the mycelium of the fungus at 96 and 144 hds (Table 1).

At the end of the trial, boscalid + pyraclostrobin, carbendazim, fluazinam, cyprodinil + fludioxonil and prochloraz inhibited to a larger extent the growth of S. sclerotiorum, in all tested concentrations and time intervals. This corroborates the findings of other researchers (^{Walter et al., 2005}; ^{Junior et al., 2008}). In another in vitro study, the carbendazim at 1 ppm inhibited at 90% the mycelial growth of the fungus mentioned (^{Martínez-Pérez, 2008}). While in the present study, carbendazim 0.25 ppm significantly reduced the development of isolated S. sclerotorium of Sinaloa. Carbendazim has been widely used in northern Sinaloa.

However, like other benzimidazoles, their effectiveness decreases when the inoculum pressure is high and the atmosphere is favourable to the fungus. In this regard, it is important to determine whether prolonged use of benzimidazoles, for over 30 years in the region, has led to the loss of sensitivity of some populations.

The in vitro inhibition of S. sclerotiorum with fluazinam is not surprising as this fungicide is one of the most effective against white mold at a dose of 0.5 L ha-1. In literature a suppression of the mycelium of this fungus, at concentrations as low as 0.1, 1 and 10 L L-1 is reported (^{Junior et al., 2008}).

For over 10 years, the carbendazim and Fluazinam are widely used against white mold in beans, in northern Sinaloa. In recent seasons the most used fungicide are boscalid + pyraclostrobin; the ciprodonil + fludioxonil and prochloraz new alternatives to chemical control of white mold in beans are represented.

Bio-rational biological effectiveness of fungicides. At 24 hds, fungal growth was zero in all treatments, except the control, the radius of the colony was 4 mm, although statistically there was no difference between treatments.

At 48 hds, the fungus was abolished in 100%, salicylic acid, hydrogen dioxide, as well as extracts from citronella, clove and grapefruit seed in two concentrations; also with the concentration of 1000 ppm of cinnamon and garlic extracts. Instead, cinnamon extract and 500 ppm concentrations of both citrus seed extract, ESC + quercetin + natural antibiotic complex (CAN), behaved similarly to the control (Table 2).

At 72, 96 and 120 hds, suppression of the fungus was maintained with both doses of salicylic acid, hydrogen dioxide, grapefruit seed extract and concentration of 1000 ppm of garlic and lemongrass. On the other hand, cinnamon extract concentration of 500 ppm and the two doses of ESC + Quercetin + CAN presented a growth similar to the control. Also, the concentration of 500 ppm of garlic extract at 96 and 120 hds also decreased the development of S. sclerotiorum compared with the control (Table 2).

At the end of the bioassay bio-rationales substances (240 hds), the two concentrations tested salicylic acid, hydrogen dioxide and grapefruit seed extract and 1000 ppm citronella extract maintained absolute control of the fungus. The effectiveness of different treatments decreased as the time passed. Garlic extract and citronella concentration of 500 ppm, and citrus extract and extract citrus seed mixture of ESC + Quercetin + CAN, in both concentrations, showed the largest colony development than the control (Table 2).

These results suggest that some compounds have the potential to control the white mold under field conditions. It is suggested to confirm the efficacy evaluating the effect of different doses, application intervals, among other tests. We also need to assess the possible phytotoxic effect. In another sense, grapefruit seed extract and citronella, may also be effective in treating fruits and vegetables after harvest, where S. sclerotiorum may be potentially important (^{Agrios, 2005}), since the freshly harvested fruits may present emerging infections, which happened a few days to manifest themselves in severe decay in storage or shelf.

Although, white mold can be controlled with synthetic fungicides, they are expensive, and the negative impact on the environment, there has been encouraged incentivized using bio-rationales substances that can replace short-term chemical-synthetic substances (^{Stauffer et al., 2000}). With regard to the above, the salicylic acid expressed as an activator of the mechanisms of systemic acquired resistance (SAR) for its acronym in plants against attack by pathogens, cold stress, UV radiation and osmotic stress (^{Conrath et al. 1995}), for this we must add that, at the tested concentrations in vitro proved fungitoxic. Hydrogen dioxide is a contact fungicide and preventive, whose broad spectrum disinfectant action includes plant pathogenic fungi and bacteria. This disinfectant (1000 ppm) controlled Fusarium oxysporum f. sp. lycopersici in tezontle and nutrient solution, which has the potential to be used in hydroponics against this disease (García-Jiménez, 2012). The grapefruit seed extract has demonstrated its ability in vitro to kill or inhibit the growth of a large amount of potentially harmful bacteria, fungi, viruses and protozoan parasites (^{Trapman, 2004}); as well as being a natural microbicide broad spectrum, a biodegradable antioxidant, nontoxic to animals and is not even corrosive. Finally, the essential oil Citronella Cymbopogon nardus (L.) Rendle with good control level S. sclerotiorum fumigant also showed activity on Aspergillus spp., Colletotrichum musae and Pyricularia grisea (^{Aguiar et al., 2014})