Introduction

The floriculture is a global industry in developing countries and developed countries; the volume of world trade is estimated at a value of more than $100 billion per year (African Bussiness Magazine, 2012). In Mexico, floriculture is an activity of economic importance, existing in the country about 2.2 thousand hectares planted gladiolus (^{Escalante et al, 2006}). The crop is affected by different types of pests and diseases, the main fungal diseases, stresses the cross rust, caused by the causative agent Uromyces transversalis (Thümen) G. Winter, same that was officially reported in Mexico in November 2004 (^{CESAVEM, 2005}; ^{Rodríguez et al, 2006}).

In the State of Mexico around 800 ha are planted; however, yield and flower quality are affected by diseases such as "cross rust" causing major losses to 90% of production (^{CESAVEM, 2005}; ^{Schubert et al, 2006}). For the management and control of this disease in the State of Mexico, have implemented various strategies based primarily on the use of synthetic chemical fungicides, favoring the emergence of resistant fungal pathogens, so new products for disease management are required (^{Vásquez et al, 2013}).

The ^{SAGARPA (2005)}; ^{CESAVEM (2005}); ^{CESVMOR (2006)}, recommend the use of fungicides as Azoxystrobin®, Trifloxystrobin + Oxycarboxin®, Amistar® 50 and Epoxiconazol®, to control the disease from first symptom detection (pustule), taking the more effective the application Oxycarboxin + Tebuconazole. In recent decades, they have increased biological studies aimed at implementing methods with potential effect on disease control, due to the recent tendency to use less harmful to the environment alternatives (^{Flores et al, 2013}). An innovative strategy for managing pests and diseases of plants of agricultural importance, is the biological control by antagonistic microorganisms to the pathogen (^{Whipps, 2001}; ^{Harman, 2006}).

Within the biological control there the use of natural enemies (introduced or manipulated), whose knowledge of its effectiveness and its mechanism of action is essential for the management and development of strategies for the implementation of sustainable agriculture (^{Torres et al., 2008}). The use of native antagonistic fungi adapted to the conditions of crop management, can give better results in reducing pathogen attack, but it is necessary to evaluate them before their recommendation. Because of this, the present study aimed to test and evaluate the efficiency antagonistic in vitro conditions, six strains of the fungus Cladosporium sp. (ClGl), Alternaria sp. (AltGl), Aspergillus sp. (AspGl) and Trichoderma sp. (TrZA, TrJi and TrPi) against the cross rust (Uromyces transversalis), as well as evaluating the rate of infection in five municipalities (Villa Guerrero, Zumpahuacan, Malinalco, Jocotitlan and Ixtlahuaca) of the floriculture area of the State of Mexico, to establish if the use of mycoparasites as drivers disease, is an effective replacement for synthetic chemical control alternative.

Materials and methods

Sampling. In the period from July to December 2013 were made 15 samples of commercial plantations with gladiolus rust (Uromyces transversalis), distributed in five municipalities that make up part of the floriculture area of the State of Mexico; Villa Guerrero, Zumpahuacan, Malinalco, Jocotitlan and Ixtlahuaca. The crop sampling took place during the stages of flowering, harvest and postharvest. The 15 sheets of each sample were collected randomly.

Assessment of severity. The 12 sheets of plants at random for each sample plot were chosen, determining the severity of the disease by a scale adapted to foliar level ^{Núñez and Romero (1980)} (Table 1).

Table 1 Severity scale for assessing the level of damage Uromyces transversalis on gladiolus leaves. 

Nivel de daño. Se calculó empleando la fórmula: P= [Σ (n * v)/ cat. mayor * N] * 100, de Towsand y Heuberger (T y H); donde: p= °/o de infección; Σ= sumatoria; n= número de plantas por categoría; v= valor numérico de cada categoría; cat. mayor= categoría mayor; N= número de plantas por muestra.

Isolation and identification of antagonistic fungi. Of infected leaves by U. transversalis on gladiolus, Puccinia horiana on chrysanthemum, Phragmidium mucronatum in rose, and leaves of carnation and chili infected apple with Cladosporium sp, were identified under stereomicroscope antagonistic fungi placed on the phytopathogenic fungi of each crop, using taxonomic keys to imperfect fungi (^{Barnett and Hunter, 1972}) and were grown in culture medium potato-dextrose agar (PDA brand Bioxon) acidified with 25% lactic acid (pH 5.5); isolates were incubated between 5-7 days at 26 °C ± 2 °C for sporulation of fungi. Because of its easy and fast growth, and its characteristics mycoparasite other fungi (^{Quiroz et al., 2008}), three strains of Trichoderma sp, isolated from tomato (TrJi), pine (TrPi) and a grass were employed oats (TrZA).

A record growth of 5 mm from each colony was taken with a sterile punch and transferred to Petri dishes with PDA to obtain uniform crops. Strains of antagonistic fungi were prepared under two dilutions from a petri dish colonized, which liquefied and capacity in distilled water at 600 and 300 mL. The obtained liquid was poured into plastic bottles with a capacity of750 mL, to which were added oil micro-refined soybean oil (EPA) and penetrating surfactant in aqueous solution (Inex-A). Also, the colony forming units (UFC) of each preparation, quantified by Neubauer were determined.

Antagonism in vitro tests. Following the modified methodology ^{Hsiang et al. (2004)}, cross-sections were performed with razor in the stereomicroscope gladiolus leaves with pustules U. transversalis approximately 3X7 cm. Each cut was deposited on a plastic base disinfected with sodium hypochlorite (NaClO). It was placed in each cut using a micropipette 0.7 uL of the suspension of each antagonist UFC on each pustules, previously counting the total number of these. Once poured drop UFC, they cut the tissue passed along with the plastic bases in Petri dishes containing agar-water acidified with lactic acid 25%, incubated for 5 days under germination chamber at a temperature of25 °C ± 2 °C. The colonization of antagonists was observed every 24 h under stereomicroscope.

Percentage colonization. The final evaluation was considered at 5 days, when the pathogen completely covered the pustule and tissue culture maintain turgor, using the formula: (%) colonization= (No. of pustules colonized/No. total pustules) X 100, formula adapted from

^{Phillips and Hayman (1970)}.

Microscopy. They mount temporary cuts of successful antagonists fungal growths on U. transversalis pustules were made. The sections were stained with cotton blue 0.5% and were photographed in a compound microscope with software Pixel Pro Version 1.1.0.

Statistic analysis. With the information of the study variables obtained, with a block model completely random variance analysis according to model that includes the design used were made (Littell et al, 1996), and when the test F analysis of variance was significant, we proceeded to perform the test means using for this, the minimum difference honest significant (DMSH) Tukey test (p≤ 0.05) (^{Palaniswamy and Palaniswamy, 2006}).

Results and discussion

Distribution and severity of disease. Assessment of severity of plantations sampled gladiolus in 15 commercial crops in the municipalities of Zumpahuacan, Jocotitlan, Malinalco, Villa Guerrero and Ixtlahuaca, a degree of infection was obtained that ranged from 22% in Villa Guerrero to 91.7% in Malinalco (Table 2). The disease was detected in the stages of flowering, harvest and postharvest, without any record in young plantations.

Table 2 Damage caused by U. transversalis on gladiolus, by municipality. 

Statistically significant effects were observed (p≤ 0.05) in the percentage of damage of the 5 municipalities evaluated, with the municipality of Malinalco presented the highest percentage of infection (Table 3).

Table 3 Percentage of infection in leaves Gladiolus by U. transversalis by municipality. 

Medias con la misma letra no son estadísticamente diferentes (Tukey, 0.05).

According to the altitude of the municipalities, it is clear the percentage difference in the degree of rust infection. While the floriculture area of Villa Guerrero occupies the largest area of cultivation for domestic and foreign sales (^{Aquino and Ríos, 2010}), it is also the municipality of increased use of fungicides, insecticides and synthetic herbicides par with best practices crop management could explain the low (%) of infection compared to other municipalities, which predate less time gladiolus introduction for production. In addition the recent introduction of the crop to the municipalities of Malinalco and Jocotitlán, as well as the use of synthetic fungicides could favor the emergence of resistant fungal pathogens, as mentioned ^{Vásquez et al. (2013)}.

In the field observations was noted that some of these plantations were poorly handled by producers. Of which it is assumed that if the disease is not combat time, can cause the total loss of the crop, such as records of ^{Schubert et al. (2006)}. ^{Aquino and Ríos (2010)} and ^{CESAVEM (2007)}, who report to Ixtlahuaca and Jocotitlan as apparently free of rust municipalities. However according to the results, these municipalities had a percentage greater than 50% infection.

Isolation and identification of antagonistic fungi. Samples of the towns of Villa Guerrero (3 strains) and Zumpahuacan (5 strains), eight isolates of antagonistic fungi associated with rust were obtained. The total fungal strains antagonists employees were ten, five strains of the genus Cladosporium sp, one of the genus Aspergillus sp, another Alternaria sp, and three of the genus Trichoderma sp. These results agree with those found by ^{García et al. (2005)}, who obtained native strains of Cladosporium, part of the leaf chrysanthemum infected Puccinia horiana. Agricultural ecosystems contain a large number and diversity of beneficial microorganisms ^{Duffy et al. (2003)}, so it is not uncommon antagonistic fungi isolated from all samples of flowers and leaves have been collected. The inhibitory effect of antagonistic fungi to pathogens, was measured with the growth of the first, from this analysis only six strains were selected to proceed with the evaluation of the antagonistic interaction in vitro (Table 4).

Table 4 Isolation of antagonistic fungi identified by host plant and sampled locality. 

The antagonists used for biocontrol of diseases are generally saprophytes, due to its ease of adaptation to the environment, its high nutrient competition against other microorganisms, versatility and easy handling. The Cladosporium fungus has been used as a biocontrol agent of white rust (Puccinia horiana) of chrysanthemum, obtaining a reduction of severity under greenhouse conditions of 41% and 84%, affecting the production and release of basidiospores (^{García et al, 2005}). The genus Trichoderma has been reported as a biocontrol agent of various pathogenic fungi, especially soil, due to its wide distribution and high host range (^{Howell, 2006}; ^{Aquino et al, 2007}; ^{Hoyos et al, 2008}; ^{Quiroz et al, 2008}).

Antagonism in vitro tests. The highly significant (p≤ 0.05) between treatments TrPi and ClGl and AspGl strains in dilutions of 300 mL were observed. The strains tested six, TrPi clearly invaded the largest number of pustules. Treatments dilution of 600 mL, were not statistically different, however AltGl, TrZA and TrPi strains showed a percentage greater than 50% colonization (Table 5).

Table 5 Percentage of colonization (biological effectiveness) of two concentrations of UFC of six antagonistic fungi on U. transverslis. 

Medias con la misma letra no son estadísticamente diferentes (Tukey, 0.05).

Although as many UFC strain Aspergillus sp. (Table 6), this did not influence their biological efficiency displayed in the percentage of colonization. The native strain AltGl equal percentage of colonization presented in both dilutions. However, bearing no differences in the percentage of colonization strains employed under a dilution of 600 mL, all strains are susceptible to be used with a percentage of average efficiency of 51.2%, and generally preparing antagonistic strains of cross rust will be more efficient at a dilution of 300 mL.

Table 6 Total colony forming units (UFC) per milliliter. 

The antagonistic capacity of some strains of fungi to plant pathogenic fungi can be defined based on the total or partial destruction of pathogen populations (^{Cook and Baker, 1983}). This ability is the result of the aggressiveness of the antagonist and the susceptibility of the pathogen (^{Paul, 1999}). Regarding the cross sections of fungal growth on ^{Sempere and Santamarina (2008)}, pustules mention that the antagonistic capacity is evident from the observed variation with respect to the selectivity of some strains of fungi. In microscopic observations of the settlement areas, the six strains tested produced variations such as dehydration, degradation and deformation of uredinia and urediospores.

The pustules cuts indicated that the six strains antagonists mycelium developed within U. transverslis pustule suggesting that act as hyperparasites fungi in uredia (Figure 1), which agrees with ^{García et al. (2005)}, with Cladosporium, who report that even if microscopic observations indicated that acts as a hyperparasite Cladosporium, not the possibility that antagonism of this fungus other mechanisms such as the production of antibiotics, enzymes and toxic metabolites involved discarded. Several of microorganisms used as biological control agents produce secondary metabolites that affect growth or germination by producing antibiotics and/or enzymes degrading cell wall (^{Assante et al, 2004}), it would be advisable to conduct research with strains that were evaluated in this investigation.

Figure 1 Observation in microscope compound invasion Trichoderma Pine (TrPi) in tissue and ureides of U. transverslis five days of incubation (40X). 

^{Spiegel and Chet (1998)}; ^{Hermosa et al. (2000)}; ^{Rey et al. (2000)}, they have described the mechanisms by which Trichoderma sp strains, displace phytopathogenic, appearing in this evaluation direct parasitism. The results suggest that AltGl, TrFCA and TrPi, strains acted as the most efficient antagonists any cross rust (U. transversalis) under laboratory conditions, however, be appropriate to determine whether in the greenhouse and field prevails his establishment and biological efficiency in various municipalities. You need to be supplemented with a multi-phase research approach that includes not only morphological characteristics but also biochemical and molecular study of the strains according to the provisions ^{Arrua et al. (2012)}.

Conclusions

The cross rust by Uromyces transversalis is distributed in cash crops gladiolus flower production five municipalities in State of Mexico with a severity of 22 to 91.7%. Moreover, antagonists fungi associated transverse rust are: Cladosporium sp., Alternaria sp. and Aspergillus sp. The in vitro inoculation of Uromyces transversalis pustules with TrPi (608X10⁶ mL¹ UFC), TrZA (371.2X10⁶ mL^-1 UFC) and AltGl (470.4X10⁶ mL^-1 UFC), isolates presented the greater colonization of pustules rust whose values according to the strains were 67.6, 62.6 and 60.2%, under controlled conditions of 25 °C ± 2 °C for five days. It was also noted that the mode of action of the antagonist's fungi was by hyperparasitism.