The state of Guanajuato has an exceptional potential for horticulture; it is among the first places in the production of 70 species of agricultural importance, some of the most outstanding of which include garlic (Allium sativum L.), lettuce (Lactuca sativa L.), and potato (Solanum tuberosum L.) (^{SIAP, 2013}).

Diseases are one of the most risk factors for the production of vegetables. In recent years, fungal diseases have caused major economic losses in the production of different vegetable species in Mexico and the rest of the workd (^{Fisher et al., 2012}). These have increased in the productive areas of the country, with Bajio as one of the zones with the highest incidence of fungal diseases (^{Montes et al., 2003}).

The resistance structures of some phythopathogenic soil fungi, such as sclerotia, can remain viable in the soil for over 20 years, such as in the case of S. cepivorum (^{Pérez et al., 2009}), originating different space and time distribution patterns as a product of reincorporation of the sclerotia and remains of diseased plants due to inadequate cultural practices carried out by farmers, as well as by natural processes. In this way, the inocula of S. cepivorum Berk., S. sclerotiorum, and S. minor can be scattered in the fields and remain latent in them, making disease control more difficult (^{Adams and Papavizas, 1986}; ^{Ibarra et al., 2010}).

Most farmers use fungicides as a disease control method, although its excessive use may lead to resistance in the pathogens, while generating toxic residues in foods and the environment, thus becoming a risk to human health (^{Fisher et al., 2012}); this has lead to a search for new fungicides, which, in some cases, are found in other organisms (biofungicides) (^{Angulo et al., 2009}). Biological control (BC) of phythopathogens presents itself as an efficient and affordable alternative, as well as risk-free in the face of numerous and increasing problems deriving from the excessive use agrochemicals (^{Agrios, 2005}). Most antagonic agents used in biological control are asprophytes, due to their adaptability to the surroundings, and their high capability of competition for nutrients with other organisms (^{Nelson, 1991}; ^{Michel et al., 2009}).

Based on the problem described above, the aim of this work was to determine the in vitro sensibility to biological control agents and isolated fungicides of S. minor, S. sclerotiorum, and S. cepivorum in plantations of garlic, bean, lettuce, potato, and radicchio.

The investigation was carried out in commercial vegetable fields in the state of Guanajuato and in the Plant Pathology Lab of the Life Sciences Division of the Irapuato-Salamanca Campus of the University of Guanajuato.

Obtaining isolates. S. minor was isolated from symptomatic lettuce (L. sativa) plants in San Miguel de Allende and Salamanca counties, Guanajuato; S. sclerotiorum was isolated from symptomatic lettuce (L. sativa) plants in Salamanca and Irapuato counties, Guanajuato; radicchio (Cichorium intybus) in Celaya municipality, Guanajuato; bean (Phaseolus vulgaris) in Guasave county, Sinaloa; and potato (S. tuberosum) in Santiago Tangamandapio municipality, Michoacán. Finally, S. cepivorum was isolated from symptomatic garlic plants (A. sativum) in Salamanca county, Guanajuato, and in Cordoba, Spain.

From each isolate, sclerotia were taken, along with part of the tissue that displayed symptoms of the disease; they were disinfected with sodium hypochlorite at 1%, and then planted in Potato Dextrose Agar (PDA) for eight days at 20±2 °C for S. minor; and at room temperature for S. sclerotiorum and S. cepivorum.

Biological control agents and fungicides. Biological control agents and fungicides evaluated (Tables 1, ² and ³ ) were applied based on commercial recommendations.

Table 1  In vitro Mycelial radial average growth (Mrag) of two S. minor isolates obtained from lettuce (L. sativa) with regard to 24 treatments with biological control products (16) and fungicides (8). 

^xEach value represents the average of both isolates. Values in each column with different letters are statistically different. Multiple comparison of averages DSH Tukey P≤0.05.

Table 2  In vitro Mycelial radial average growth (Mrag) of five S. sclerotiorum isolates, obtained from lettuce (L. sativa), bean (P. vulgaris), potato (S. tuberosum), and radicchio (Cichorium sp.) plants with regard to 24 treatments with biological control products (16) and fungicides (8). 

^xEach value represents the average of two isolates. Values in each column with different letters are statistically different. Multiple. Comparison of averages DSH Tukey P≤0.05.

Table 3  In vitro Mycelial radial average growth (Cprm) vitro of two S. cepivorum isolates obtained from plantations of garlic (A. sativum) with 24 treatments of biological products (16) and fungicides (8). 

^xEach value represents the average of two isolates. Values in each column with different letters are statistically different. Multiple Comparison of averages DSH Tukey P≤0.05.

In vitro sensibility and evaluation of biological products and fungicides. We weighed the commercially recommended doses of the biological products and fungicides to be evaluated and added to the agar, then poured into Petri dishes. When the agar solidified, disks, 1 cm in diameter were placed in the center of the dish from the periphery of the colonies obtained from each isolation and incubated at 20±2°C.

Evaluation of mycelium growth. The colony diameter was measured in two directions - the longest and shorteste length - and mycelial growth was obtained as the average of both values in the directions at 24, 48, 72, 96, 120, 144, 168, 192, 216, 240, and 268 hours after placing the disk with the fungi on the edge of each Petri dish in each of the three treatment repetitions (^{Pérez et al., 1997}; ^{Pérez et al., 2009}).

Statistical analysis. In the three experiments, a split plot experimental design was used, with a factorial arrangement correction with three repetitions. In each experiment there was a factorial arrangement: factor A was assigned to fungal isolations, which had two, five, and two levels; factor B was assigned to the biological control agents and fungicides, which had 25 levels. This gave a total of 50, 125, and 50 treatments, respectively. In all three experiments, the multiple comparison of averages was carried out using Tukey's test (P≤0.05).

Sclerotinia minor. The two isolates of S. minor were sensitive to seven of the eight fungicides (Dicloran, Benomyl, Boscalid, Iprodione, Tebuconazole, Cyprodinil-Fludioxonil, and Chlorothalonil), considering this as those which did not develop a mycelial radial average growth (Mrag), 264 hours after the confrontation (Table 1). The biological control agents with the greatest fungistatic effects towards S. minor isolates, that is, those which caused the least Mrag 264 hours after confrontation, were: Microrganisms (BPG-Plus), Trichoderma sp. (Trichoderma), T. viride (Esporalis) and Bacillus subtillis (Serenade max), with 1.16a, 1.31a, 1.51a, and1.85a, respectively; in comparison to Gliocadium virens (Soil gard), B. subtillis (Probac bs), Bacillus sp.-Glomus sp. (Soil cure) and B. subtillis (Natubacs), with 8.50h, 7.68gh, 6.71efg, and 7.61fgh, respectively, which had the lowest or null fungistatic effects towards the isolates (Table 1).

Sclerotinia sclerotiorum. The five isolates sensitive to four of the eight fungicides (Dicloran, Benomyl, Tebuconazole, and Cyprodinil-Fludioxonil) i.e., those which showed no Mrag 264 hours after confrontation (Table 2). The biological control agents that had the greatest fungistatic effects towards the isolates, understood as those which caused the lowest Mrag 264 hours after confrontation were Trichoderma sp. (Trichoderma), Trichoderma harzianum (Natucontrol), Microorganismos (BPG-Plus), and T. harzianum (Biotricho-H), with 1.48abc, 1.56abc, 2.35bc, and 2.53cd, respectively, in comparison with T. harzianum (Triko root), B. subtillis (Probac bs), G. virens (Soil gard), and Streptomyces lydicus (Actinovate), with 7.12h, 6.79gh, 6.80gh, and 6.32fgh, respectively, which had the lowest or null fungistatic effects towards the S. sclerotiorum isolates (Table 2).

Sclerotium cepivorum. Both isolates were sensitive to the eight fungicides (Dicloran, Benomyl, Boscalid, Iprodione, Chlorothalonil-Cymoxanil, Cyprodinil-Fludioxonil, Tebuconazole, and Clorotalonil), therefore they did not present Mrag 264 hours after confrontation (Table 3). The biological control agents that had a greater fungistatic effect on the S. cepivorum, which means that they caused the least Mrag 264 hours after confrontation, were Trichoderma sp., (Trichoderma), T. viride (Esporalis), T. harzianum (Natucontrol), and Microorganisms (BPG-plus), with 1.03a, 1.73ab, 2.55b, and 2.70bc, respectively; in comparison with G. virens (Soil gard), Bacillus sp.-Glomus sp. (Soil cure), and B. subtillis (Natubacs), with 8.50i, 8.45i, and 8.50i, respectively, which led to a lower or null fungistatic effect on the isolates (Table 3).

A uniform behavior was found in S. cepivorum towards fungicides Tebuconazole (Tebucur), Iprodione (Rovral), Dicloran (Botran), Benomyl (Blindaje 50), Boscalid (Cabrio), Clorotalonil (Trevanil), Clorotalonil+Cymoxanil (Strike), and Ciprodinilo+Fludioxonilo (Swish). Contrasting results were found in the behavior of S. cepivorum towards the fungicides reported by ^{Pérez et al. (1997)} and ^{Pérez et al. (2009)}, who showed the presence of variability of the pathogen towards the fungicides used. In relation to the response of S. cepivorum towards the biological control agents evaluated, a wide variability was found, observing that the biological products to which there was a higher sensitivity to in vitro were those formed with Trichoderma spp.; the cause of this greater benefitial effect may be the biocontrolling action of Trichoderma spp., in which there have been several forms of action described that regulate the development of phythopathogenic fungi. Among these, the main ones are the competition for space and nutrients, microparasitism and antibiosis, which have a direct action on the phythopathogenic fungi (^{Infante et al., 2009}); it is also known that Trichoderma spp. displays other mechanisms, the bioregulatory action of which is indirect, such as the biochemical defense mechanisms, that consist in the deactivation of enzymes of the phythopathogenic fungi during the infection process (^{Infante et al., 2009}); this could explain the high inhibitory percentage of mycelial growth of S. cepivorum. This suggests an alternative for the control of white rotting, as pointed out by ^{Ulacio et al. (2011}). The use of T. harzianum as an alternative of biological control in soils that display high densities of S. cepivorum inocula, requires high doses of the antagonist combined with other alternatives to achieve an efficient control of the disease (^{Delgadillo et al., 2002}; ^{Ulacio et al., 2011}). A uniform response was found in S. minor and S. sclerotiorum towards most fungicides and isolates evaluated. The isolates of radicchio from Celaya and lettuce from Irapuato grew in the presence of some fungicides, in comparison to the other isolates of both fungi that did not, probably due to the treatments undergone by the fields the isolates were taken from, since these lands are treated with biological controllers, and this may have caused the isolates to have a more vigorous mycelial growth with some of the fungicides evaluated. Something that was unexpected in this study was that the isolates of S. sclerotiorum provenientes from Celaya and from Irapuato grew in the presence of Boscalid (Cabrio), which is a fungicide that belongs to the group of Carboxanilides, and Iprodione (Rovral), which belongs to the Dicarboximide, in which there was Mrag between 24 and 264 hours after confrontation; this is probably related, among other things, to the history of the fields the S. sclerotiorum isolates were obtained from, regarding the continuous use of fungicides for the control of root diseases in different crops (^{Pérez et al., 2009}). The insensitivity to Boscalid and Iprodione obtained in this study contrasts with the sensitivity reported by ^{Tarazona (2009)}, who found that the Boscalid and Iprodione inhibited fungal growth by 100 %. The sensitivity presented by isolates S. minor and S. sclerotiorum towards Tebuconazole, which brought about an inhibitory effect of 100 %, coincides with reports by ^{Pérez et al. (1997)}, who claim these were similar with S. cepivorum. The biological control agents which caused the most inhibition of mycelial growth in S. minor and S. sclerotiorum were those that contained the fungus Trichoderma spp. The resuls obtained in this study coincide with those by ^{Villalta et al. (2012)}, who report a strain of T. hamatum with a high percentage of inhibition of the growth of S. minor. In the case of B. subtillis (Serenade max), there were no favorable effects on S. minor and S. sclerotiorum, in comparison with the favorable effect obtained with Trichoderma spp., since B. subtillis (Serenade max) was one of the biological control agents with the lowest fungistatic effect on the S. minor and S. sclerotiorum isolates. These results contrast with those reported by ^{Tarazona (2009)}, who points out that B. subtillis had a greater controlling effect towards the phythopathogenic fungi analyzed.