Biological material

Festival variety strawberry plants were acquired in the company named Insumos Agrícolas para Invernadero INAPI, in Irapuato, Guanajuato.

The bacterial strains (Pseudomonas tolassi, B. pumilus and Paenibacillus sp.) and the Arbuscular Mycorrhizal Fungus (R. intraradices) were provided by the Soil Microbiology Area, from the Soil Science Graduate Program, Colegio de Postgraduados, Campus Montecillo.

Phytophthora capsici strain PcT17 used in this experiment was provided by the Universidad Michoacana in San Nicolás de Hidalgo, Mexico.

Pathogenicity of Phytophthora capsici strain PcT17 on strawberry

Eight Festival variety strawberry plants with three and four leaves were inoculated (8 d after transplant) with 200 000 zoospores per plant, to show the pathogenicity of the oomycete in this variety of strawberry under greenhouse conditions.

First phase: Antagonistic capacity in vitro of three strains of de rhizobacteria towards Phytophthora capsici

The first phase was a test for antagonism by confrontation in vitro of the rhizobacteria of P. tolassi, B. pumilus, and Paenibacillus sp. against P. capsici. The rhizobacteria were cultivated in nutritive agar for 48 h at 27 °C and the strain P. capsici was developed in V8 medium V8 (juice of eight vegetables: calcium carbonate: agar) at 27 °C for 10 d.

Test in vitro

The mycelial growth of P. capsici in vitro was evaluated using the dual cultivation technique described by ^{Landa et al. (1997)}. V8 medium discs, 5 mm in diameter, with the active growth of the oomycete, were placed in the center of a Petri dish with V8 medium. At the same time, the bacteria were planted individually in a straight line, 3 cm away from the disc with mycelial content. The cultures were incubated for 5 d at 28 °C. The control was a 5 mm disc with V8 medium with mycelial growth, but without bacterial inoculant.

Treatments and experimental design

The experiment had four treatments, 1) P. toolassi confronted with P. capsici, 2) B. pumilus confronted with P. capsici, 3) Paenibacillus sp. confronted with P. capsici and 4) Control. The experimental design was totally at random and each treatment had four repetitions. The experimental unit was one Petri dish.

The percentage of inhibition was calculated using the following formula (^{Landa et al., 1997}):

where r is the radius of P. capsici and R is the maximum radius of the oomycete without the bacteria.

Second phase: Test under controlled environment conditions

Uniformly sized Festival variety strawberry plants were disinfected with sodium hypochlorite at 1 % for 3 min and rinsed three times with sterile distilled water. Each plant was moved to a 400 cm³ container, which was the experimental unit.

The substrate was a mixture of peat moss, agrolite and vermicompost (4:4:2 v/v/v), sterilized in an autoclave at 120 °C for 2 h, twice. The plants were watered with sterile water as required and each week a Long Asthon nutritive solution was added, modified to apply 11 mg of P L^-1 (^{Hewitt, 1966}).

The strawberry plants were inoculated 8 d after transplanting with 0.5 g of sorghum roots colonized in 80 to 85 % by R. intraradices, and 1 mL of the bacterial suspension composed of P. toolassi, B. pumilus and Paenibacillus sp. at a concentration of 108 UFC mL^-1. Strawberry plants with and without inoculant were placed in a controlled environment chamber (Sherer^® Model CEL 37-14, Gillet Marshall Mich. USA), at a constant temperature of 26 °C.

Multiplication and inoculation of Phytophthora capsici

P. capsici cultures with 10 d of growth in V8 medium were added 10 mL of sodium cloride solution at 0.9 % (Abbott^MR) per Petri dish for 10 min. The isotonic solution was poured, divided into four parts, and each was placed in a sterile Petri dish. Later, sterile distilled water was added and the P. capsici cultures were placed under cold white light at 26 °C for 48 h, and at 28 °C in the dark for 24 h.

The Petri dishes with P. capsici were placed for 30 min at 4 °C, then for 30 min at 28 °C to promote the release of zoospores. The content of each Petri dish was emptied into a container and the number of zoospores was counted using a cytometer (Marienfeld^®). The zoospore suspension was adjusted to 200 000 zoospores mL^-1.

The oomycete was inoculated in strawberry plants 45 d after the inoculation of bio-controlling microorganisms, adding 1 mL of the suspension in each container. Inoculated plants were kept for 5 d in moisture-saturated soil to promote the displacement of the oomycete. This experiment was performed twice.

Disease incidence and severity

The incidence (total number of plants wilted by the pathogen in regard to the total expressed as a percentage) and severity of the disease was registered on days 5, 10, 15 and 20 after the pathogen inoculation. Severity was evaluated using the modified scale by ^{Adorada et al., (2000)}, that assigns values from 1 to 50 according to the degree of plant wilting: 1=0 % wilted plants; 2=1-25 % of wilted plants; 3=26-50 % of wilted plants; 4=51-75 % of wilted plants; 5=76-100 % of wilted plants or death.

The disease severity (DS) was expressed as a proportion (percentage) according to the formula by ^{Adorada et al., (2000)}:

Foliar area, dry biomass weight of the aerial part, radical volume, and number of stolons

The foliar area was registered directly using a foliar area meter (Area Meter, Model LI-3100; Nebraska, U.S.A.). The weight of the dry biomass of the aerial part was determined by drying the samples in an oven (FELISA, Model 242-A) at 70 °C for 72 h y and then weighed using an analytical scale (Acculab, Model ALC-104). The volume of the root was evaluated by water displacement, submerging the root system in a graduated 100 mL test tube. The number of stolons was determined in each treatment. All variables were evaluated at the end of the experiment.

Determination of nitrogen (N) and phosphorous (P)

N and P were determined in the Soil Fertility lab of the Colegio de Postgraduados. Nitrogen, using the semi-microkjeldahl method (^{Bremner, 1975}) and phosphorous, using photocolorimetry by reduction with Molibdo-vanadate.

Determination of mycorrhizal colonization

Mycorrhizal colonization was determined using the root staining and clearing method (^{Phillips and Hayman, 1970}).

The frequency of the mycorrhizal colonization in strawberry plant roots was determined using the method by ^{Biermann and Linderman (1981)} and was expressed as a percentage.

Experimental design and treatments

The experimental design was totally at random, with eight treatments and six repetitions each. The treatments were: 1) Negative control (not inoculated), 2) Positive control (inoculated only with P. capsici), 3) Plants inoculated with PGPR’s (Pseudomonas tolassi, Bacillus pumilus, and Paenibacillus sp.), 4) Plants inoculated with PGPR’s+P. capsici, 5) Plants inoculated with R. intraradices, 6) Plants inoculated with R. intraradices+P. capsici, 7) Plants inoculated with PGPR’s+R. intraradices and 8) Plants inoculated with PGPR’s+R. intraradices+P. capsici. The experimental unit was a 400 cm³ pot with one plant.

An ANOVA was performed using the data, the means of the treatments were compared using Tukey’s test (p>0.05), and SAS Version 9.0 (SAS Institute, 2002) was used.

First phase: Antagonistic capacity in vitro of P. tolassi, B. pumillus and Paenibacillus sp. towards Phytophthora capsici

The three bacterial strains had an antagonistic effect against P. capsici (Table 1 and Figure 1). P. tolassi and B. pumillus inhibited the growth of the oomycete by 54 %, and Paenibacillus sp. by 43 %. These results suggest that the rhizobacteria probably produce secondary metabolites that spread throughout the agar and inhibit the growth of the phytopathogen.

Figure 1 Inhibition of Phytophthora capsici by rhizobacteria. A) P. capsici growing alone (Control), (B) P. capsici with Bacillus pumilus (C) P. capsici with Pseudomonas tolassi and (D) P. capsici with Paenibacillus sp. 

^{Landa et al. (1997)} report that some strains of Pseudomonas produce secondary metabolites that spread in agar, causing the inhibition of Fusarium spp. Also, some Bacillus species exert an antagonistic effect due to the production of lytic enzymes, antibiotics or metabolites, or both, which produce changes in the cytoplasmic membrane (^{Sicuia et al., 2015}; ^{Ramyabharathi and Raguchander, 2014}).

Second phase: Test in controlled environment

Disease incidence and severity

On days 0 and 3 no incidence of the disease was observed. Plants inoculated only with the pathogen showed 83 % incidence of the plant at the end of the experiment. However, plants pre-inoculated with PGPR’s or R. intraradices showed incidences of 50 and 33 %, respectively. The manifestation of the disease in plants inoculated with PGPR’s+R. intraradices was 33 % (Figure 2).

Figure 2 Disease incidence caused by Phytophthora capsici in strawberry plants pre-inoculated with Rhizophagus intraradices and PGPR’s. The incidence was registered on days 5, 10, 15, and 20 after inoculationg with the pathogenic oomycete. Each point corresponds to the average of six plants. 

The severity of the disease began manifesting itself in plants not inoculated with beneficial microorganisms, five days after the inoculation of the pathogen. At the end of the experiment, the severity of the disease in positive control plants was 63 %, yet plants pre-inoculated with PGPR’s showed a severity of 37 % (Figure 3), due possibly to the induction of systemic resistance (^{Nadeem et al., 2014}), or when competing for nutrients and niche (^{Ochoa et al., 2010}).

Figure 3 Disease severity caused by Phythophthora capsici in strawberry plants pre-inoculated with Rhizophagus intraradices and PGPR’s. The severity was registered on days 5, 10, 15, and 20 after inoculation with the pathogenic oomycete. Each point corresponds to the average of six plants. 

Plants inoculated with R. intraradices displayed a severity of 33 %. ^{Alarcón et al. (2000)} indicate that apart from the nutritional benefit of the symbiosis, AMFs also participated as root pathogens biological control agents.

Plants inoculated with PGPR’s+R. intraradices presented a lower severity (23 %) (Figure 3). AMFs and rhizobacteria act syngergically, stimulating plant growth and the inhibition of the pathogenic oomycete (Figure 4). PGPR’s stimulate AMF growth (^{Linderman, 1993}), root development, a greater susceptibility of the root to the colonization of mycorrhizal fungi, and improves the process of recognition between roots and fungi (^{Artursson et al., 2006}; ^{Bonfante and Anca, 2009}).

Figure 4.Response of the Festival commercial variety strawberry premycorrhized with Rhizophagus intraradices and inoculated with PGPR’s to the attack of Phytophthora capsici. Non-inoculated plants (T1), plants inoculated with P. capsici (T2), plants inoculated with only PGPR’s (T3), plants inoculated with PGPR’s+ P. capsici (T4), plants inoculated with only R. intraradices (T5), plants inoculated with R. intraradices + P. capsici (T6), plants inoculated with R. intraradices +PGPR’s (T7) and plants inoculated with R. intraradices, PGPR’s+ P. capsici (T8). 

PGPR’s suppress diseases caused by pathogenic microorganisms with the production of siderophores, synthesis of antibiotics, enzymes, or fungicidal compounds or both (^{Lugtenberg and Kamilova, 2009}). For example, Pseudomonas and Bacillus can control pathogens, particularly fungi, synthesizing antifungal molecules (^{Whipps, 2001}). Bacteria of the genus Pseudomonas induce plant resistance by increasing the speed and levels of synthesis of phytoalexins, substances directly implied in the plant’s defense (^{Lemanceau and Alabouvette, 1993}).

Foliar area, dry biomass weight of the aerial part, radical volume, and number of stolons

Plans inoculated with PGPR’s+R. intraradices showed greater foliar areas (239.95 cm²), dry biomass weight (6.24 g) and root volume (2.1 mL) (Table 2). These results coincide with reports by ^{Vosatka et al. (2000)} and ^{Linderman (1993)}, who state that the use of joint mutualistic symbionts (nitrogen fixers in free life such as Pseudomonas and Bacillus) affects the increase in plant growth and production. Also, the symbiotic-mutualistic relationship in the roots of fruit-producing plants leads to diverse physiological changes or modifications and highlights the increase in photosynthetic activity due to the greater CO₂ fixation capacity (^{Alarcón and Ferrera-Cerrato, 2000}); therefore, the rates of dry biomass produced increase (Alarcón et al., 2000). According to ^{Díaz et al. (2013)}, the symbiosis with AMF provides benefits in the host plant, such as increase in growth and mineral nutrition.

Plant growth and nutrition (Table 2) was improved by the rhizobacterial production of growth regulators, as reported by ^{Molina-Romero et al. (2015}). Plants inoculated with PGPR’s+R. intraradices produced five stolons, plants inoculated with R. intraradices produced three, and plants inoculated with PGPR’s, only one. This result may be due to the synthesis of growth regulators such as gibberellins and cytokinins (^{Alarcón et al., 2000}). In the other treatments there was no stolon production.

Nitrogen (N) and phosphorous (P) contents

The concentration of N and P in the foliage was not different (p>0.05) between treatments (Table 3), although PGPR’s promote plant growth by directly providing N, P, and essential minerals (^{Ahemad and Kibret, 2014}). PGPR’s can fix atmospheric nitrogen due to the presence of the enzyme nitrogenase, making this N accessible to plants with which they associate (^{Gupta et al., 2015}). The three species of PGPR’s used in our study (B. pumilus, P. tolassi, and Paenibacillus sp.) are reported as non-symbiotic N-fixating bacteria (^{Hernández et al., 2009}; ^{Glick et al., 2007}; ^{Fernandes et al., 2014}). There were no significant differences in the concentration of N in the foliage and the highest value (1400 mg kg^-1) was observed in plants inoculated only with PGPR’s. The non-symbiotic, N-fixating PGPR’s only provide small amounts of the N fixated onto the host plant (^{Glick, 2012}). Also, the low contribution of N by bacteria is affected by environmental conditions, such as high concentrations of O₂ in the soil atmosphere, which inhibit nitrogenase (^{Alquéres et al., 2010}), or high concentrations of H₂ and NO that compete with the substrate of nitrogenase (^{Hichri et al., 2015}; ^{Gabrielyan et al., 2015}). The inoculation of HMA and PGPR’s did not increase (p>0.05) the P in the foliage due to its high reactivity with Fe, Al, and Ca and to its resulting precipitation. The ranges of precipitation may be up to 90 % (^{Adesemoye et al., 2008}); likewise, the soil and the environmental conditions impact the efficiency of PGPR’s and HMAs (^{Adesemoye et al., 2008}).

The highest growth rate was registered in plants inoculated with HMA+PGPR’s. Genera Pseudomonas, Azotobacter, and Bacillus release indole3-acetic acid (AIA), gibberellins or cytokinins in the rhizosphere of plants and they exert a growthstimulating effect, particularly in the plantlet stage (^{Lugtenberg and Kamilova, 2009}). Also, some PGPR’s produce enzymes that favor plant growth and development with the reduction of ethylene. Such bacteria take ACC, the precursor of ethyene, and turn it into 2 oxobutanoate and NH₃ (^{Glick et al., 2007}).

Mycorrhizal colonization

For total mycorrhizal colonization there was no statistical differences between treatments. The highest numerical value (70 %) was registered in the treatment of plants premycorrhized with R. intraradices and inoculated with PGPR’s, and the lowest (39.75 %), in plants inoculated with R. intraradices+P. capsici (Table 4). ^{Frey-Klett et al. (2007)} report the involvement of different genera of soil PGPR’s in the mychorrhizal colonization process; this group of PGPR’s is known as mycorrhization helper bacteria. The highest percentages of total colonization, vesicles and hyphae in our experiment were obtained when PGPR’s were inoculated (Table 4). Probably, these bacteria promoted mycorrhizal colonization, but not sporulation in strawberry plants.

Vesicles were the predominant structures and the number was the same in plants inoculated with R. intraradices and plants inoculated with PGPR’s+R. intraradices (Figure 5).

Figure 5.Vesicles (A) and spores (B) found in strawberry plant roots inoculated with Rhizophagus intraradices and PGPR’s (100X y 40X, respectively).