Problems Found in the Recent Bonus System 1 Problematic bonus formula

Salinas-Castro A1*, San Martín-Romero E2 & Suárez-Medellín J3 1

Facultad de Ciencias Agricolas Universidad Veracruzana, Lomas del Estadio S/N Zona Universitaria , Xalapa, Veracruz, México; [email protected]

2

Laboratorio de Alta Tecnología de Xalapa, S.C., Médicos No. 5 Unidad del Valle Xalapa, Veracruz, México; [email protected]

3

Centro de Investigaciones Cerebrales, Blvrd Adolfo Ruiz Cortines, U.H. del Bosque, 91010 Xalapa, Veracruz, México; [email protected]

* [email protected]

ABSTRACT

Potato (Solanum tuberosum L.) is one of the most important crops worldwide. Unfortunately, due its high demand, usually requires a great amount of agrochemicals, which are often known for not being environmentally or health friendly. This work is aimed to evaluate in vitro the ability to phosphate solubilization, nitrogen fixation and siderophore production of several bacterial strains belonging to genus Serratia and Pseudomonas, obtained from vigorous greenhouse potato crops, free of agrochemicals and pathogens. A set of biochemical tests were carried out on Providencia alcalifaciens, Pseudomonas syringae pv. syringae, Serratia marcescens, Serratia liquefaciens I, Serratia liquefaciens II, Pseudomonas putida and Serratia ficaria, which were previously identified by PCR. Mineral phosphate solubilizing activities of all strains were tested on tricalcium phosphate medium; for siderophore production it was used NaCl 0.85% medium; for the nitrogen assay it was used biological fixation medium (NFB) as well as Kjeldahl digestion of the inoculated plant material. The results obtained showed significative differences in phosphate solubilization for S. ficaria, followed by Pseudomonas putida and S. liquefaciens I. The only species that presented siderophores

52 was S. ficaria. In the nitrogen fixation assay, the plants irrigated with the strain P. putida showed the highest average nitrogen contents (2.8% from dry weight), followed by P. syrige pv. siringe and S. liquefaciens I (2.7% and 2.4%, respectively). In regards of the in vitro experiment, all seven strains showed positive results. It was concluded that the rhizobacteria studied exhibit several plant growth promoting features, such as phosphate solubilization, nitrogen fixation and siderophore production, particularly the strains Serratia ficaria, S. liquefaciens I, Pseudomonas putida and P. syringae pv. syringae, which suggests that their use in agriculture might be a promising environmentaly friendly strategy.

Key words: Bacteria, Nitrogen fixation, Siderophores, Phosphate solubilization.

There are around 10 millon microbial species found in soil, acting both as decomposers and organic compounds mineralizers. About of millon species of bacteria exists in every gram of soil from the rhizosphere, which might exert a positive or negative influence on the plants (Rico-Gallegos, 2009; Reyes et al., 2008; García–Pineda et al., 2014). One of the positive effects exerted by some rhizobacteria is the growth promotion of different crops by direct and indirect mechanisms, such as nitrogen fixation, siderophore production and phosphate solubilization, among many others (Ahemad & Kibret, 2014). On the other hand, soil pollution caused by the indiscriminated use of agrochemicals, has triggered a growing interest in the search of novel environmentaly friendly alternatives, in order to mitigate the damage in the soil quality to attain better crop yields (Reyes et al., 2008; Beltrán-Pineda, 2014).

Countries like Brazil or Colombia are pioneers in the use of bacterial inoculants in the agriculture, which has lead them to important savings in production cost by diminishing or even suppresing the aplication of sinthetic fertilizers (Estrada et al., 2012).

One of the most important crops worldwide is potato (Solanum tuberosum L.). In Mexico, this tuber is found all year long, from sea level to heights above 2,400 m.a.s.l. The main producer states are: Sinaloa, Sonora, México, Chihuahua and Veracruz states, which report a volume of 920,829 tons, representing 75% of national production. Nevertheless, potato is also one of the most agrochemical-demanding crops (Sinfuentes-Ibarra et al.,

53 2013; SIAP 2016). The goal of this research was to evaluate in vitro the ability of bacterial strains belonging to the genera Serratia and Pseudomonas to solubilizate phosphate, fixate nitrogen and produce siderophores, as an alternative to be used in potato crops.

MATERIALS AND METHODS

Bacterial were isolated from the rhizosphere of potato crop, cultivar Perote, from El Conejo, Veracruz, México 97.15 N and 19.53 W and 3140 m in elevation. The bacterial strains were identified by sequencing of DNA as: Providencia alcalifaciens (PRAL), Pseudomonas syringae pv. syringae, (PSSY) Serratia marcescens(SEMA), Serratia liquefaciens I (SELI-I), Serratia liquefaciens II(SELI-II), Pseudomonas putida (PSPU) and Serratia ficaria (SEFI), as previously reported by Salinas-Castro et al., 2016.

The bacterial strains were subjected to in vitro tests for phosphate solubilization, siderophore production and nitrogen fixation. A nitrogen percentage evaluation for potato plants (Solanum tuberosum L.) periodically irrigated with bacterial solutions, was also performed.

Phosphate solubilization: In order to evaluate the phosphate solubilization caused for each of the studied strains, it was employed the tricalcium phosphate medium reported by Shekhar in 1999. In this assay there were used bacterial suspensions at a concentration of 1,8x109 in Mc Farland scale.

Three Petri dishes with specific culture medium for phosphate solubilization, each one with three sterile 500µ filter paper discs were inoculated with bacterial suspensions of each strain, and then incubated at 28°C for twelve days. Then, the halos produced by each bacterial strain were observed and its diameter was measured in milimeters. An ANOVA and a Tukey test to compare the means were carried on using a significance level of 0.05. All statistical analysis were performed with R software v3.2.1 (R Core Team, 2015). Fluorescent siderophore production: A bacterial suspension of each strain was performed in NaCl 0.85%. Then 10 μL alicuotes of the suspension were taken and cultured by triplicate on a Petri dish with Chrome azurol S agar (CAS), using one microdrop per dish. The plaques were incubated for 48 h at 30±2 °C. Positive results were observed by the turn of yellow to blue around the bacterial growth (Louden et al. 2011).

54 Nitrogen percentage of potato plants (Solanum tuberosum L.): For this assay it was used the potato cultivar “Fianna”, which is one of the most highly demanded for national consume market. 21 tubers were sprinkled with 250 ml of a bacterial suspension containing a cell concentration of 1.2 x 108. One month after the sowing in green house conditions, three reinforcement aplications of the bacterial suspension were performed to each experimental unit, once every two weeks. The plants were kept under irrigation conditions during a three months period, and then foliar samples were taken to perform the analysis of nitrogen percentage, both dry and wet basis.

Nitrogen percentage was derermined by Huldych (2008) 2g of dry and grounded leaf samples from potato plants were obtained from each treatment and replication. The samples were placed in a Kjeldahl tube, and 0.3g of selenium mix and 5ml of sulphuric acid were added. Kjeldahl digestion was carried out at 360°C by about 3 h and then cooled until the acid vapors emision stoped and crystals were observed. samples were distiled using a BÜCHI 339 apparatus, and the nitrogen percentage was determined according the following formula:

Nitrogen %= (V1-V2) x N x 0.014 x 100 W (g)

Biochemical test for nitrogen fixation: All seven strains studied were inoculated in tubes with semiliquid nitrgen fixation medium (NFB), leaving the negative control without inoculum. The tubes were incubated during two weeks at 30ºC. The positive reaction was manifested as a halo-like bacterial growth (Döbereiner et al. 1999).

55 RESULTS

Phosphate solubilization: All seven bacterial strains showed ability to solubilizate phosphate. However, the only strain that presented a significant difference acording the ANOVA and the Tukey test was S. ficaria, which showed a 20 mm halo, followed by S. liquefaciens with a halo of 18mm. Finally, the rest of the strains P. putida P. syringae, S. marcescens, S.liquefaciens I and P. alcalifaciens presented some phosphate solubilization, even though, their halos were smaller (Figures 1 and 2).

Figure 1. Tricalcium phosphate solubilization diameter of seven bacterial isolated from potato roots (p ≤ 0.05).

56 Figure 2. Phosphate solubilization halos displayed by Serratia ficaria (SEFI),

Pseudomonas putida (PSPU), and Serratia liquefaciens II (SELI-II).

Siderophore production: From all seven strains evaluated in this studio, only S. ficaria showed fluorescence under UV light, demonstrating the siderophore production in vitro, as shown in Figure 3.

Total nitrogen percentage in potato plants: From the bacterial suspension sprinkled to potato plants, the strain that generated a greater mean nitrogen percentage on its host was Pseudomonas putida with 2.8% on dry basis, followed by P. syringae with 2.7% and S. ficaria with 2.6% of nitrogen respectively. On the other hand, S. marcescens showed the smallest mean nitrogen percentage with 2.1% (See Table 1). In regard of the biochemical nitrogen fixation test, all seven strains presented a positive reaction (+), since all of them presented a bacterial growth halo, as shown in Figure 4.

Figure 3. Siderophore production, determined by fluorescence of S. ficaria (SEFI) in NaCl 0.85% culture medium.

TABLE 1 Total nitrogen percentage obtained in dry and wet weight basis from potato plants irrigated with the different bacterial suspensions

Bacterial strain % Nitrogen dry weight basis % Nitrogen wet weight basis

Pseudomonas putida 2.8 0.5 P. syringae 2.7 0.4 Serratia ficaria 2.6 0.4 Providencia alcalifaciens 2.5 0.5 Serratia liquefaciens I 2.4 0.3 S. liquefaciens I 2.4 0.4 Serratia marcescens 2.1 0.3

57 Figure 4. Biochemical nitrogen fixation test in semi liquid culture medium (NFB), where is shown the negative control ( - ) as well as the seven strains studied.

DISCUSSION

The ability of some rhizosphere bacteria to solubilizate phosphate, allow them to be considered as plant growth promoters. Bacteria that shows this feacture in vitro is expected to facilitate the absorption of this nutrient by the plant (Reyes & Valduz, 2006; Kumar et al., 2000 y Mrkovacki & Milic, 2001). There are several examples, that stand for the benefits of the application of phosphate-solubilizing bacteria (PSB) on crops of economic importance, such as the study reported by Sánchez-López and coworkers, in which tomatoe plants (Solanum lycopersicum L.) were inoculated with Pseudomonas putida PSO14 and Enterobacter sp. TVL-2 in greenhouse conditions, reaching yield increments between 17 % and 49 % Sánchez-López et al., 2012). Meanwhile, in this work carried on with bacteria isolated from the potato crop rhizosphere, it was observed that the species belonging to genera Pseudomonas and Serratia showed the ability for phosphate solubilization.

On the other hand, it has been reported that several fungi and bacteria are able to produce litic enzymes in order to exert biocontrol on their host. One of the main bacterial species with that feature is Serratia marcescens, which has also been included in this work (Soler- Arango, 2012).

Regarding nitrogen fixation, all seven bacterial strains studied, presented growth halos that indicate a positive response to this test. Similarly, there is a growing wealth of knowledge suggesting the nitrogen fixation capacity of Pseudomonas sp in different crops

58 and environments (Orozco-Jaramillo & Martínez-Nieto, 2009), which is also true for several other bacterial species belonging to the genera Azospirillum, Enterobacter, Klebsiella, Pseudomonas and Burkholderia (Estrada et al., 2001).

The genus that has more reports of exerting Plant Growth Promoter Bacteria (PGPR) activity is Pseudomonas spp, which has been related to indirect effects such as control of pathogen microorganisms, production of antifungal and antibiotic compounds, phosphate solubilization, siderophores production and sistemic resistence induction (Dowling & O`Gara 1994; Keel & Défago 1997; Guevara-Avendaño, 2015; Adame-García et al., 2016). In this study, the only strain capable of generate siderophores was Serratia ficaria. Nitrogen fixation is an energeticaly expensive process for bacteria, although it represents a major competitive advantage for them, so the whole process depends mainly in the disponibility of such element in the soil (Orozco-Jaramillo & Martínez-Nieto, 2009), which might explain the reason for the relatively low amounts of total nitrogen (2.1 to 2.8%) in plants treated with bacterial solutions, found in this study. Probably the soil in which the experiment was carried on did not present enough disponibility of such element (Rivacoba-Gómez, 2013).

According to the aforementioned reasons, in this work is concluded that all the bacteria studied, showed desirable features for plant-growth promoting bacteria, such as: the ability for phosphate solubilization, nitrogen fixing, and siderophore producing in vitro, particularly the strains Serratia ficaria, S. liquefaciens I, Pseudomonas putida and P. syringae pv. syringae, which makes them a suitable option to be used agriculture with an environmentaly friendly approach.

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ANEXOS

Alejandro Salinas Castro:

Thank you for submitting the manuscript, "Biochemical properties of bacterial species isolated from potato crop rhizosphere" to Revista de Biología Tropical/International Journal of Tropical Biology and Conservation. With the online journal management system that we are using, you will be able to track its progress through the editorial process by logging in to the journal web site: Manuscript URL: http://revistas.ucr.ac.cr/index.php/rbt/author/submission/27539 Username: asalinas32 If you have any questions, please contact me. Thank you for considering this journal as a venue for your work.

Julián Monge Nájera

Anexo

Artículos publicados producto de la estancia en el LATEX

Annals of the Entomological Society of America Advance Access published August 26, 2016

Annals of the Entomological Society of America, 2016, 1–5

doi: 10.1093/aesa/saw057 Research article

Arthropods in Relation to Plant Diseases

Stethobaroides

nudiventris

(Coleoptera:

Curculionidae), the Curculionid Cause of Petal

Wilting on the Catasetum integerrimum Orchid

M. Morales-Ba´ez,1 A. Salinas-Castro,1,2,3 D. E. Bello,2 M. G. L. Cadena,1 A. R. Ferna´ndez,2 and A. Trigos1,4

1Laboratory High Technology Xalapa, University Veracruzana, Medical 5, Forest Unit, Xalapa-Veracruz 91010, Mexico (angel [email protected]; [email protected]; [email protected]; [email protected]), 2Faculty of Agricultural Sciences, University Veracruzana, Circuit Gonzalo Aguirre Beltr´an s/n, Isleta, Xalapa-Veracruz, CP 91090, Mexico (d.estevez.bello@gmail. com; [email protected]), 3Corresponding author, e-mail: [email protected], and 4Center for Biomedical Research, University Veracruzana, Av. Doctor Luis Castelazo Ayala s/n, Industrial Animas, Xalapa-Veracruz, CP 91190, Mexico

Received 19 May 2016; Accepted 19 July 2016

Abstract

The baridinos have gained importance in agriculture; however, many of them are yet to be described, as is the case for those affecting orchids. It is for this reason that the aim of this research was to identify the insect causing the wilting of petals on orchids belonging to the Catasetum integerrimum species at a site at La Estanzuela, municipality of Emiliano Zapata, and El Espinal, municipality of Naolinco, in the state of Veracruz, Mexico. Orchids of the species C. integerrimum in specimens with flowers affected were monitored, as insects cause damage to these at the time of flowering. Thus, according to the morphological characteristics of larvae and adults and of the male and female genitals observed in

vegetative material, as well as larvae and adults present on the flowers, it was confirmed that the species of curculionid that attacks the orchid is Stethobaroides nudi- ventris Champion, 1907. This is the first report of this curculionid as the agent causing wilting on C. integerri- mum petals.