Increase in Fusarium oxysporum f. sp. lycopersici (R1), Alternaria solani and Botritys cinerea

The first stage started with the inoculum increase. According to the protocol established by ^{Cañedo and Ames (2004)}, the fungus was obtained from the infected material, either by sowing pieces of potato-dextrose-agar (PDA).

The identification for each fungus was made according to the ^{Jarvis monograph (1975)}, in addition to the keys of ‘CMI descriptions of pathogenic fungi and bacteria’ belonging to the Commonwealth Micological Institute. In the same way, the support of the keys for identification of fungi was used by ^{Barnett and Hunter (1998)}; ^{Gilman (1963)}; ^{Romero (1993)}. To have the fungi with the minimum genetic variation, monosporic cultures of all the strains were made, from which the sample was taken.

The suspensions of conidia in tubes were stored under refrigeration at 4 °C until use. Subsequently, this material were performed in the pathogenicity tests seed for Fusarium oxysporum f. sp. lycopersici (R1)= (Folr1), was developed in tomato vegetative material Bonny Best and Manapal (without resistance genes and resistant to Folr1, respectively), for A. solani was in leaves and B. cinerea in fruit being made in the variety ‘Río Grande’ whose characteristics is that it is of a certain type and fruit type saladette and susceptible to both phytopathogenic agents.

The pathogenicity tests consisted in an inoculation of Folr1 in differential materials; for the case of seed, 20 seeds were imbibed in 200 ml in a conidial suspension (Folr1) of 10⁸ conidia ml^-1 and the seeds were seeded in unicel cups (½ liter) containing sterile peat-moss substrate (Sunshine, Sun Gro Horticulture Canada, Ltd.); likewise, it was carried out in seedlings with a development of 20 days after sowing, through a conidial suspension of 10⁸ ml^-1 conidia through the immersion of roots to which small wounds were previously made with a hypodermic needle.

They were immediately transplanted into ½ liter beaker vessels containing the same peat-moss type sterile substrate (^{Rueda et al., 2006}). The plants were maintained at an ambient temperature of approximately 30 ±2 °C. Four repetitions of a plant in each material were used for the data collection, the response of the differential materials was observed and recorded to ensure also and autoinfection by Folr1. The irrigation was carried out with sterile distilled water, according to the technical recommendations (^{INIFAP, 2005}). Wilt symptoms in inoculated seedlings were presented 30 days after inoculation. The evaluation was made again based on the presence or absence of the disease.

Regarding the tests on leaves and fruits, the first ones were inoculated with A. solani with the help of a hand sprayer-sprinkler, of 250 ml, while the fruit was inoculated with B. cinerea by first puncturing them and adding with a 1 ml cotton swab of conidial suspension at a concentration of 10⁸ ml^-1. After the inoculations, the organs were placed in humid chambers at a temperature of 30 ±2 °C in a period of 30 days (^{Jarvis and Hargreaves, 1973}; ^{Jarvis, 1975}; ^{Davised, 1988}; ^{Eckert, 1988}; ^{Tello and Lacasa, 1988}; ^{SAGARPA, 2005}), which are appropriate conditions to induce the signs of the disease. In each of the tests, a control was included (negative control - use of sterile water). Confirmation of the pathogens was carried out using the Elisa serological technique following the general protocol of identification of fungi AGDIA.

Production of antiserum

The immunization was carried out in rabbits of the New Zealand breed with a weight of 3 kg, an age of 9-24 months and avoiding that the females were pregnant (Kirali, 1974; ^{Bokx, 1980}; ^{Valdes, 1995}; ^{Villarreal, 1980}). The immunization plan was carried out in three schemes, with five rabbits per scheme (Table 1), according to the methodology recommended by ^{Flores (1994)}; ^{Rueda et al. (2006)}.

Obtaining imported tomato seed that is planted in the state of Sonora

To obtain the seed, we had the collaboration of the National Confederation of Horticultural Producers (CNPH) Sonora region, where each producer provided an amount of 100-150 seeds for the analysis.

Sampling in plants (seedling, developed leaf and fruit)

For the sampling of lots, the national potato sampling carried out by the ^{SARH (1994)} was reproduced. Sampling was carried out on 10% of the total arable land of three of the tomato producing municipalities (Table 2). In 10% of the surface of each region divisions of 5 ha were made that would be considered as a hypothetical plot to be sampled. At each point an imaginary diagonal line was drawn from corner to corner, and 10 samples were collected on that line, the samples collected, previously identified, were wrapped with moist paper and placed in a cooler to be transferred to the laboratory for analysis.

Detection of Fusarium oxysporum f. sp. lycopersici (R1), Alternaria solani and Botritys cinerea in seed of import in culture media

According to the technique of ^{Randhawa (1996)} and ^{Rueda et al. (2006)}, each sample of seed was washed in tap water for 30 min in order to eliminate chemicals (ie. fungicides) and placed in plastic trays with a capacity of 2 L. Each tray with the seed was added 2 ml of phosphatase buffer solution with a pH= 7. The mixture of water with phosphatase containing each seed sample was called ‘mother suspension’, the trays were incubated for 12 h in refrigeration at 4 °C in order to be released conidia of Folr1, A. solani and B. cinerea to the mother suspension.

After incubation, 10 ml of mother suspension was taken from each of the trays, four dilutions were made to such suspension (10:1, 10:2, 10:3, 10:4) and the last dilution was took 0.1 ml that was seeded in PDA culture medium in Petri dishes by the rod dispersion method (^{Roger et al., 1981}). The media were incubated for seven days at 34 °C. Once the conidia were germinated and there was mycelial growth with reproductive structures, these were identified considering the keys indicated in the phase of increase of the strains.

Detection of Fusarium oxysporum f. sp. lycopersici (R1), Alternaria solani and Botritys cinerea in seed, seedling, developed leaf and fruit with the antiserum produced

The seedlings, developed leaves and sampled fruits were subjected by a process of cuts of 0.5 to 1 cm in diameter and introduced directly into a saline solution at 0.85% NaCl, called the mother solution. For the detection of Folr1, A. solani and B. cinerea, the slide agglutination technique was developed as indicated by ^{Kiraly (1974)}.

Detection of Fusarium oxysporum f. sp. lycopersici (R1), Alternaria solani and Botritys cinerea in seed, seedling, developed leaf and fruit by Elisa technique

For the detection of Folr1, A. solani and B. cinerea, with respect to the Elisa serological technique, the protocol described in seed detection was considered.

Pathogenicity tests of those samples positive for Fusarium oxysporum f. sp. lycopersici (R1), Alternaria solani and Botritys cinerea, with different detection methods

For the reaffirmation of the fungi Folr1, A. solani and B. cinerea that turned out to be positive in the previous detection methods, the pathogenicity tests were carried out using the technique of ^{Randhawa (1996)} and ^{Rueda et al. (2006)}, as described in the purification and increase of inoculum to produce antigen.

Identification tests

When carrying out the previous tests of inoculum increase, and identification (specific culture media and ELISA) and of pathogenicity, the result was the same as those indicated by ^{Jarvis (1973)}; ^{Cañedo and Ames (2004)}; ^{Ascencio-Álvarez et al. (2008)}; López (2012), State Committee for Plant Health of Guanajuato, AC (^{CESAVEG, 2016}); ^{Robles-Carrion et al. (2014)}; ^{Valencia et al. (2016a)}, indicating that for Folr1, the characteristics are to produce colonies of fast growth, they present an aerial mycelium, cottony and of white coloration; microscopically they show macroconidia, fusiform, curves at the ends and septate; the dimensions of the macro and microconidia (29.1-45 X 2.9-4.7 μm), the development of conidia was observed at nine days, item that agrees with ^{Valencia et al. (2016b)}.

In relation to the fungus Alternaria solani, the development of conidia was achieved after 8 days; result that agrees with ^{Cazar et al. (2014)}, this presented a cottony aerial mycelium, which soon turned black when sporulated. The conidiophores are dark measuring 12-20 X 120-296 μm, dark, with the appearance of a mallet and have longitudinal and transverse septa (^{Martínez-Ruiz et al., 2016)}.

On the other hand, Botrytis cinerea, presented gray colonies. On the fifth day, well-differentiated, simple, straight conidiophores of 205-210 x 18 -20 μm, branches of 18-20 x 6-8 μm were observed. Ellipsoidal, smooth, almost hyaline conidia, 7-14 x 5-8 μm, often with a 3 μm long appendage according to ^{Farrera et al. (2007)}; ^{Robles-Carrion et al. (2014)}.

Regarding pathogenicity tests, the symptoms of seeds embedded in the conidial suspension of 10⁸ ml^-1 conidia, with Folr1, 100% germinated between 36 and 48 h under favorable conditions of the disease in both materials used (Bonny Best and Manapal), the seedlings after 18 days showed a slight wilting on the stem. The cotyledons showed irregular spots with a darker and more greasy appearance in relation to the healthy area. This same symptomatology was identified in seedlings that once dead were observed as the fungus fructified on the surface of the stem under humid conditions. The opposite occurred with the Manapal material, where the manifestation of symptoms was not detected or when developing trans and horizontal cuts along the conductive tissues in root and seedling stem. The results obtained agree when carrying out inoculations in the same material Bonny Best and Manapal, with ^{Ascencio-Álvarez et al. (2008)}.

For Alternaria solani are the symptoms on leaves, at nine days those were characteristic circular, close to 1.5 cm in diameter, brown containing concentric rings. The results agree with ^{Martínez-Ruiz et al. (2016)}, indicating that the first symptoms appear on the oldest leaves and progress towards the newer leaves; from there you can also see brown spots on the pedicels and on the chalices when they are attached to the flower or fruit. In this way, the fruits become infected; through the calyx or the pedicel both when they are green or mature.

For Botritys cinerea the symptomatology began with the formation of small concentric rings; the first odors of putrefaction were detected and on the 13th day there were already conidiospores. The results obtained agree with ^{Martínez-Ruiz et al. (2016)}.

Through confirmation by the Elisa serological technique, a positive result was obtained for the fungal specimens provided by the donor company, as well as suspensions of cellular juice and infected tissue rich in conidia obtained from the aforementioned pathogenicity tests.

Antigen production against Fusarium oxysporum f. sp. lycopersici (R1), Alternaria solani and Botritys cinerea

Because not all warm-blooded animals react as an antigen, only Scheme 3 (Table 1) was found to be efficient to produce a high antibody content. The agglutination was reaffirmed by making readings under the compound microscope to observe a microagglutination with the help of a compound microscope (Table 2) (^{Bokx, 1980}).

Detection of Fusarium oxysporum f. sp. lycopersici (R1), Alternaria solani and Botritys cinerea in the state of Sonora

In the case of seed, the results are shown in Table 2. Under the technique of Diagnosis using solid means papa-dextrosaagar (PDA), the presence of Folr1 in the seed donated by producers belonging to the Coast of Hermosillo region was demonstrated. Valley of Guaymas and Yaqui, with the materials DMX1 and DMX2, results similar to those indicated by ^{Valencia et al. (2016}a), ^{Robles-Carrion et al. (2014)}. The same result occurred in the seed obtained from Valley of Yaqui in the BER and GLO material, but with the difference that they turned out to be positive for Alternaria solani, since the observed conidia corresponded to that according to ^{Robles-Carrion et al. (2014)} and ^{Martínez-Ruiz et al. (2016)}.

With respect to the Elisa technique, the results indicate that, by this technique, the seed obtained from the three regions with the DMX1 and DMX2 materials turned out to be positive to the presence of Folr1, being confirmed with the samples processed with respect to the control.

With respect to the agglutination and microagglutination technique, the results were positive for Folr1, in seed samples obtained from producers in the sampled regions (DMX1 and DMX2, BER and GLO), with the exception of the Guaymas region with RUE, ZAP materials. , LEO and DAR that also turned out to be negative. In Table 2, the results vary only for the pathogen A. solani, where the diagnostic technique with the antiserum produced is detected in the DMX1 and DMX2 materials, the opposite occurred in the Elisa, hence the importance of the difference of results and to define if those samples presented or not conidial cells belonging to the fungi studied (^{Rueda et al., 2006}).

Sampling of seedling, developed leaf and fruit in commercial lots

Regarding the study of seedling, leaf and fruit made by means of the PDA culture medium technique, the Elisa technique and antiserum produced for the detection of Folr1, A. solani and B. cinerea, the results are shown in Table 3. In can appreciate that the vegetative materials of the three sampled regions that were found to be positive to the presence of Folr1, in seed, when detected in seedling or in the subsequent phenological stages, the result was negative; this may be due to the fact that the varieties that are sown are resistant to Folr1 or because the cultural and management conditions, as well as those within the production areas, such as high temperature and low atmospheric humidity, could cause the pathogen does not have the ability to survive and therefore not produce an infection in the tomato plant. Alternaria solani and Botritys cinerea under the techniques carried out, vary among the techniques; it can be seen that the technique of Elisa and Antiserum produced, were sensitive to the presence of these two phytopathogens, otherwise the PDA medium occurred (Table 3).

Tests of pathogenicity to positive fungi in vegetative samples by the three methods of detection

These tests were only carried out on those samples obtained from the sampling sites and which turned out to be positive in the diagnostic techniques used. Pathogenicity tests showed that, when inoculated into seed, 20-day-old seedlings, leaves and fruit, seed and seedling for Fusarium oxysporum f. sp. lycopersici (R1), leaf for Alternaria solani and fruit for Botritys cinerea with a respective positive and negative control, under favorable conditions of the disease, turned out to be positive according to the characteristics described by ^{Ascencio-Álvarez et al. (2008)}, in vegetative material Bonny Best and Manapal. On the other hand, A. solani was in leaves and B. cinerea in fruit being made in the variety ‘Rio Grande’.