**2. Material and methods**

#### **2.1. Detection of brown rot (***Ralstonia solanacearum***) of potato crop (***Solanum tuberosum* **L.) of the state of Sonora, Mexico**

The research was carried out in two stages: the first one consisted in the increase of *R. solanacearum* (*Rs*) with specific culture media, tuber pathogenicity tests, seedling and fruit for familiarization purposes in *Rs*, and their identification by ELISA technique. The second stage included a field sampled of tuber, seedling, flowering, and tubers produced in physiological maturity plants in Sonora state, to detect *Rs* by specific culture media and ELISA. Likewise, pathogenicity tests were carried out on those samples that were positive for the presence of *Rs*. Fit in mention that Detection of *Rs* in potato tuber was carried out in two types of tuber: (a) those from Canada and the United States of America (USA) and (b) tuber which it is to consumption human and used as a seed, it was sampled in commercial stores in Sonora state.

#### *2.1.1. First stage*

prevent their dispersion from primary inoculum sources, the negative aspects mentioned above have, to a certain degree, caused losses and major disruptions in some regions to national agriculture, so it is advisable to have the necessary measures to prevent the entry and secondly the control and dispersion of these biological agents harmful to plants [1]. In the Mexican Republic, specifically in northwestern Mexico, the area targeted for the cultivation of vegetables (mainly tomato, potato, chili, and watermelon) has increased considerably in recent years [2]. Mexico does not produce quality seed, it obliges producers to acquire tuber of foreign origin, mainly from the United States, since the varieties acquired produce fruits that meet the characteristics preferred by the consumer and also cause the tuber volumes to enter the country which is a gateway for microorganisms of quarantine importance such as *Pseudomonas solanacearum* = (currently) *Ralstonia solanacearum* to potato, *Xanthomonas campestris* pv. *vesicatoria* to chili and tomato, *Clavibacter michiganensis* ssp. *sepedonicus* to potato, *C. michiganensis* spp. *michiganensis* to tomato, and *Acidovorax avenae* pv. *citrulli* to watermelon, among others [1]. Also, the controversy is currently being generated among producers about their presence in agricultural fields in the state of Sonora, Mexico. Therefore, it is of great importance to know the current situation of *R. solanacearum* in potato (*Solanum tuberosum* L.), on material and during the different phenological

This research aims to expand knowledge of the situation that occurs in the detection of bacteria of quarantine importance, in addition to updating and reaffirming its null presence in the agricultural areas of the state, and later extend to the interior of the country. In addition, this screening study is aimed at involving producers with a new production scheme under

On the other hand, in recent years, there has been a growing interest in the use of biologically active compounds extracted from plant species that have the ability to eliminate pathogenic microorganisms by themselves, mainly due to the resistance that microorganisms have developed to antibiotics [3]. In addition, agriculture in the new millennium must establish new control alternatives that produce a lower environmental impact, as day-to-day increases in the percentage of consumers who demand healthy and chemical-free food [4]. Therefore, the importance of knowing new control strategies arises, especially those that have a sustainable aspect. Based on the above, the need to evaluate bactericidal products to perform tests of anti-

According to the abovementioned details, the principal goals were to detect the presence of *R. solanacearum* in tuber and vegetative material of *S. tuberosum* L. and to evaluate the bacteri-

The research was carried out in two stages: the first one consisted in the increase of *R. solanacearum* (*Rs*) with specific culture media, tuber pathogenicity tests, seedling and fruit for familiarization

**2.1. Detection of brown rot (***Ralstonia solanacearum***) of potato crop (***Solanum* 

stages of the crop in the agricultural zone of the state of Sonora, Mexico.

phytosanitary conditions in order to safeguard our national agriculture.

microbial activity against *R. solanacearum* is presented.

cidal effect of essential oils.

126 Potato - From Incas to All Over the World

**2. Material and methods**

*tuberosum* **L.) of the state of Sonora, Mexico**

It was initiated with the increase of *Rs* and was developed according to the protocol established by Rueda [1], using means of specific cultures. According to the technique described by the same author, ten test tubes were obtained in 0.85% NaCl saline solution with a concentration of 108 colony-forming units (CFU)/ml, verified with the aid of a hematometer. Bacterial suspensions in tubes were stored in refrigeration at 4°C to stabilize the bacterium and avoid a shock in the immunization.

Pathogenicity tests (PPs) were developed in order to familiarize themselves with the symptomatology of *Rs* which were carried out in tuber and potato seedlings. In the case of tuber, 20 tubers were split in half and inoculated into the bundle by 200 ml with a bacterial suspension of 108 CFU/ml, and were placed in humid chambers under favorable conditions of the disease; likewise, another 20 tubers were considered as negative control when going through the same process but making use of sterile distilled water in the incision. Regarding PP in seedlings, 40 tubers were previously disinfected and were germinated in germination plates with sterile substrate, the conditions in which the seedling produced was at 25°C using sterile tap water for irrigation; at the end of 30 days after the emergency, 20 seedlings were inoculated with the bacterial solution of *Rs* at a concentration of 108 CFU/ml with the aid of a cotton swab on the cotyledons of the seedlings, and the remaining 20 seedlings were considered as negative control when passing through the same process with swab plus sterile distilled water. The tubers, seedlings used for PP, after inoculation, were covered with polyethylene bags and placed in an incubation chamber with a relative humidity between 80 and 90% and a temperature of 35–41°C in a period of 4–7 days [1]; these conditions are appropriate to induce the signs of the disease.

For the process of identification of *Rs*, the serological ELISA technique was developed, following the general protocol of identification of bacteria AGDIA. The *Rs* kit was donated by the project to which this research belongs.

#### *2.1.2. Second stage*

In the second stage, farmers donated tubers from Canada and the USA; the tuber, which is consumed by human and used as a seed, was sampled in commercial stores in Sonora state. Also, fields of potato crop were sampled in three stages: seedling, flowering stage, and physiological maturity when the plants produced tubers considered to be cut and later for sale. It should be noted that seedlings, leaves, or fruits of plants showing a symptomatology similar to that of *Rs* were also collected. Batch sampling was according to the National Potato Sampling in Mexico [8]. Sampling was carried out in 10% of the total cultivated area of nine municipalities of Sonora state (Agua Prieta, Caborca, Cajeme, Hermosillo, Moctezuma, Navojoa, Sahuaripa, Santa Ana, and Ures). Located in the geographical coordinates, Agua Prieta 31° 17′ north latitude and 109° 33′ west longitude, Caborca 30° 42′ north latitude and 112° 09′ longitude west, Cajeme 27° 29′ north latitude and 109° 56′ longitude west, Hermosillo 29° 05′ north latitude and 110° 57′ longitude west, Moctezuma 29° 47′ north latitude and 109° 40′ west longitude, Navojoa 27° 03′ north latitude and 109° 25′ west longitude, Sahuaripa 29° 03′ north latitude and 109° 14′ west longitude, Santa Ana 30° 33′ north latitude and 111° 07′ west longitude, and Ures 29° 25′ north latitude and 110° 23′ west longitude. In the 10% of the surface of each municipality, the following was done: each batch of 5 ha was considered as a sampling area. Each hectare of that surface was a must-see. At each point an imaginary diagonal line was drawn from corner to corner, and on that straight line, ten samples were collected. Donations from producers were obtained. Each of the collected samples, previously identified, was wrapped with wet paper and placed in a cooler to be transferred to the laboratory for analysis.

Research and Postgraduate in Food of the University of Sonora, in the city of Hermosillo, Sonora, Mexico. The experiment was carried out in vitro under controlled conditions at 30 °C

*Ralstonia solanacearum*: A Bacterial Disease and Its Biological Control by Essential Oils on *Solanum tuberosum* L.

http://dx.doi.org/10.5772/intechopen.70744

129

The bacterial strain used in the study was *R. solanacearum*, was isolated, and characterized

The bacterial strain was grown in a culture of 24 hours at 30°C in Nutrient Broth (Difco, Sparks, MD) (extract of 3.0 g and peptone of 5.0 g) and adjusted to a concentration of 108 CFU/ml with phosphate-buffered saline (PBS). The bacterial inoculum was massively planted on dextrose and potato agar plates using a sterile cotton swab to achieve uniform microbial growth [6].

Once the plates were inoculated with the bacteria, filter paper disks of approximately 10 mm in diameter were placed in the center of the dish, in which different amounts of the essential

Essential oils were prepared at different concentrations using 70% ethyl alcohol as diluent. The concentrations used were 1:1, 1:5, and 1:10. Aseptically, 7.5, 10, and 15 μl of each of the concentrations of the essential oils were placed on the filter paper disks. Seventy percent alcohol was used in one of the filter paper disks as a negative control to discard the antimicrobial activity of the same. In addition, a disk of streptomycin (10 μg/disk) and one ampicillin (10 μg/disk) were used as the reference control. After impregnating the disks with the respective treatment, the plates were incubated at 30°C for 24 hours. After the incubation period, bacterial growth inhibition halos were measured in millimeters using a ruler. Analyses were

The experimental design was trifactorial A × B × C where factor A has two oils [oregano (*Lippia graveolens*) and thyme (*Thymus vulgaris*)], factor B has three dilutions (1:1, 1:5, and 1:10), and factor C has three amounts applied 7.5, 10, and 15μl. The data were analyzed in Statistix 8.0 program (2003).

When testing for pathogenicity for *R. solanacearum* (*Rs*) symptoms in potato seedlings, the results indicate that between the tubers embedded in the bacterial suspension of 108 CFU/ml and between 7 and 15 days under favorable conditions of the disease, the vascular bundle of the tuber was darkened, and, when making a cross section, a grayish bacterial mucilage was exuded by the eyes and by the end of the stolon in the tubers. There were grayish-white outcrops that exudate from the darkened vascular ring of the cut tubers. On the other hand, in the inoculated seedlings when making a transverse cut at the stem level, the exudation of a graybrown mucilage was noted. This could be verified by makng a transverse cut at the base of the

**3.1. Detection of brown rot (***Ralstonia solanacearum***) of potato crop (***Solanum* 

from pathogenicity tests of potatoes from commercial houses in the state of Sonora.

and 90% humedity.

oils were applied.

carried out in triplicate.

*tuberosum* **L.) of the state of Sonora (Mexico)**

**3. Results**

*3.1.1. First stage*

Detection of Rs in tuber. According to Rueda [1], each tuber sample, consisting of 10 tubers from each batch of cooperating producers, weighed separately, washed in running water for 30 min, and placed in plastic trays with a capacity of 2 L. Each tray, with its respective sample of tuber, is left with an amount of 100 ml of distilled water, and each of these trays was added 2 ml of buffer solution phosphatase with a pH = 7. The water-phosphatase mixture containing each tuber sample is called a "mother suspension." The trays were incubated for 12 hours in cooling at 4°C in order to release the bacteria to the stock suspension. After the incubation, 10 ml was taken from suspension of each of the trays, four dilutions were made to such suspension (10:1, 10:2, 10:3, 10:4), and the last dilution of 0.1 ml was taken and sowed in specific culture medium in Petri dishes by the rod dispersion method The media were incubated for 7 days at 34°C. The inoculated media were then incubated at a temperature of 35°C for 7 days [1].

Detection of Rs in vegatitive material sampled (tuber, sedling, leaf and fruit), by ELISA technique. For the detection of *Rs*, with respect to the serological ELISA technique, the protocol described in the detection of the microorganism in the PP was considered.

Detection of *Rs* in tuber, seedling, leaf developed and fruit by PCR technique. Commercial primers were obtained from the 16Sr intergenic region, and screening tests were performed to reaffirm the null or positive presence.

Pathogenicity tests to positive bacteria with the different methods of detection. For the reaffirmation of *Rs* bacteria that proved to be positive in previous detection methods, they were carrying out pathogenicity tests [5]. The PPs were applied to seedlings 25–30 days after emergence, as described above in the PP of the first stage. The diseased tissue bacteria were reinoculated using the Randhawa technique, and the pathogen was confirmed by ELISA.

#### **2.2. Evaluation of the in vitro antibacterial activity of essential oils of oregano and thyme against** *Ralstonia solanacearum*

This stage which consisted of the evaluation of the antibacterial activity of two essential oils was carried out in the laboratory microbiology and mycotoxins of the Department of Research and Postgraduate in Food of the University of Sonora, in the city of Hermosillo, Sonora, Mexico. The experiment was carried out in vitro under controlled conditions at 30 °C and 90% humedity.

The bacterial strain used in the study was *R. solanacearum*, was isolated, and characterized from pathogenicity tests of potatoes from commercial houses in the state of Sonora.

The bacterial strain was grown in a culture of 24 hours at 30°C in Nutrient Broth (Difco, Sparks, MD) (extract of 3.0 g and peptone of 5.0 g) and adjusted to a concentration of 108 CFU/ml with phosphate-buffered saline (PBS). The bacterial inoculum was massively planted on dextrose and potato agar plates using a sterile cotton swab to achieve uniform microbial growth [6].

Once the plates were inoculated with the bacteria, filter paper disks of approximately 10 mm in diameter were placed in the center of the dish, in which different amounts of the essential oils were applied.

Essential oils were prepared at different concentrations using 70% ethyl alcohol as diluent. The concentrations used were 1:1, 1:5, and 1:10. Aseptically, 7.5, 10, and 15 μl of each of the concentrations of the essential oils were placed on the filter paper disks. Seventy percent alcohol was used in one of the filter paper disks as a negative control to discard the antimicrobial activity of the same. In addition, a disk of streptomycin (10 μg/disk) and one ampicillin (10 μg/disk) were used as the reference control. After impregnating the disks with the respective treatment, the plates were incubated at 30°C for 24 hours. After the incubation period, bacterial growth inhibition halos were measured in millimeters using a ruler. Analyses were carried out in triplicate.

The experimental design was trifactorial A × B × C where factor A has two oils [oregano (*Lippia graveolens*) and thyme (*Thymus vulgaris*)], factor B has three dilutions (1:1, 1:5, and 1:10), and factor C has three amounts applied 7.5, 10, and 15μl. The data were analyzed in Statistix 8.0 program (2003).
