Antagonism tests in vitro

Dual confrontations were performed in order to evaluate the antagonistic effect of Saccharicola sp. A trial with seven treatments was established: 1) Antagonist-Phytophthora capsici (A-Pc), 2) Antagonist-Fusarium oxysporum (A-Fo), 3) Antagonist-Rhizoctonia solani (A-Rs), 4) Antagonist control (A), 5) Phytophthora capsici control (Pc), 6) Fusarium oxysporum control (Fo) and 7) Rhizoctonia solani control (Rs). Each treatment included 10 replications. In each Petri dish 90 mm in diameter filled with Potato-Sugar-Agar Infusion (IPAA), a cylinder 5 mm in diameter containing mycelium of the antagonist was placed 1 cm from the periphery and then kept in darkness at 27±1 °C. Four days later, a cylinder 5 mm in diameter filled with IPAA containing Pc, Fo or Rs mycelium was placed at the opposite end of the dish. A cylinder filled with Pc, Fo or Rs was placed at each end of the control dishes. The dishes were kept under the conditions previously mentioned.

Evaluated variables

Mycelial growth in the different treatments was measured (in cm) every 24 h and the number of days when hyphae of the confronted isolates came into contact was recorded for each treatment. When the mycelium of both the antagonist and the pathogen came into contact, the growth reduction percentage of the phytopathogen was calculated using the formula proposed by ^{Sid-Ahmed et al. (1999)}. For this calculation we considered the growth average (in cm) of the pathogens in the control dishes, as well as the growth average from the dual confrontation.

The dishes were kept under observation during 30 days to record the number of days when the antagonist developed on the pathogens’ mycelium. The viability of the mycelium of the pathogens confronted with the antagonist was also evaluated when it developed on the pathogens colony. When the antagonist developed on P. capsici, F. oxysporum and R. solani colonies, five Petri dishes were taken and from each dish three cylinders containing medium and showing mycelial growth of the invaded phytopathogen, and sown in IPAA to determine whether Pc, Fo and Rs mycelium was viable. After sowing, observations were made every 24 h during 5 days to determine if the phytopathogen was developing.

To know the antagonist effect on phytopathogen propagule production (zoospores or spores) at 21 dpc for P. capsici, and at 30 dpc for F. oxysporum, the following procedure was used: for F. oxysporum, from five dishes containing A-Fo treatments and their corresponding control (Fo), three cylinders 5 mm in diameter filled with mycelium were taken from the side where the pathogen developed and placed in tubes (Sarstedt^®) filled with 5 ml of sterile distilled water; then the cylinders were shaken, and, using a micropipette, 200 µl of suspension were taken to count micronidia using a hemacytometer (Marienfeld^®).

For P. capsici, from each of the five Petri dishes, three cylinders 1 cm in diameter filled with mycelial growth were taken and placed in dishes 50 mm in diameter filled with sterile distilled water and incubated at 27±1 °C for 48 h under white light to induce sporangia formation. After this period, and to induce zoospore release, the dishes were kept at 4 °C for 20 min, and then at 27±1 °C for 1 h. Observations were made under a stereoscopic microscope to record the relative abundance of zoospores in relation to that of the control.

Mycelial interaction between Saccharicola sp-P. capsici and Saccharicola sp-R. solani

To know if the Saccharicola sp antagonist infects P. capsici and R. solani mycelia, microcultures were prepared following the technique described by ^{Riddell (1950)} with slight modifications. The experiment consisted of five treatments, and observations were made to four replications every 24 h during 15 days with a total of 60 Petri dishes for each treatment. The evaluated treatments were: 1) Antagonist-P. capsici (A-Pc); 2) Antagonist-R. solani (A-Rs); 3) antagonist control; 4) Pc control; and 5) Rs control.

Five mm cylinders containing the antagonist mycelium were sown in Petri dishes with IPAA at 1 cm from the periphery. A previously sterilized 18 x 18 mm cover slip was taken with a sterile tweezer and placed in the middle of each dish. After 72 and 96 h, a 5 mm cylinder containing Pc or Rs mycelium was placed at a distance of 1 cm. In all cases, the dishes were kept in darkness at 27±1 °C. The same procedure was used for the controls. Every 24 h during 15 days observations were made to cover slips in four replications of each treatment; when mycelium of both microorganisms was found in the middle of the cover slip, it was removed from the culture medium using a sterile razor. A drop of cotton blue was added to the cover slip and observed under a 100 X Olimpus® model CX31 compound microscope.

Scanning electron microscopy (MEB): The analyzed samples were taken from the interaction areas of confrontation between the antagonist and the pathogen (P. capsici) in IPAA medium. The samples were analyzed at the Red de Estudios Moleculares Avanzados, Instituto de Ecología A. C., Cluster BioMimic^®, Xalapa, Veracruz, Mexico.

Antagonism tests in greenhouse: A trial was established from November 2015 to January 2016 using soil coming from the field and collected in a plot naturally infested with P. capsici. One portion of the soil was sterilized (SE) twice (2 h each) in an autoclave at 65 °C and 20 lb pressure, and then ventilated during 7 days. The other portion was not sterilized (SN). Eleven treatments were tested using a random blocks design with 10 pots (a jalapeño pepper plant of the “M” variety in each pot filled with 500 g of soil).

The treatments used were: 1) Control (SE-T), 2) SE-F. oxysporum (SE-Fo), 3) SE-P. capsici (SE-Pc), 4) SE-R. solani (SE-Rs), 5) SE-Antagonist (SE-A), 6) SE-Antagonist-F. oxysporum (SE-A-Fo), 7) SE-Antagonist-P. capsici (SE-A-Pc), 8) SE-Antagonist-R. solani (SE-A-Rs), 9) SE-Antagonist-F. oxysporum-P. capsici-R. solani (SE-A-Fo-Pc-Rs), 10) SN-Antagonist-F. oxysporum-P. capsici-R. solani (SN-A-Fo-Pc-Rs), and 11) SN-F. oxysporum-P. capsici-R. solani (SN-Fo-Pc-Rs). As reference of the pathogenicity of the P. capsici isolate used, chili plants of the highly susceptible variety “Yollow wonder” were also inoculated. The trial was repeated one more time.

Seed germination

Seeds of the jalapeño pepper M variety were disinfected with 2 % commercial sodium hypochlorite for 3 min and rinsed three times with sterile distilled water. Seeds from the treatments including the antagonist were introduced in a 1X10⁸ colony-forming unit (ufc) of Saccharicola sp suspension; the other treatments were kept in sterile running water. The seeds were placed in sterile paper towels (sanitas) and left to germinate in darkness at 27±1 °C for 11 days.

Seedlings were transferred to polyestirene trays containing a previously sterilized mixture of Peat Mos and Agrolita (2:1). To the seedlings from the treatments including the antagonist a 1X10⁸ ufc suspension (10 ml/seedling) was added immediately after being transferred to the trays. The trays were kept in a growth room with photoperiods of 14 h light and 10 h in darkness at 25±1 °C until seedlings developed the first pair of true leaves.

Preparing antagonist and pathogen inoculum, and inoculation.

Inoculum of the antagonist and of R. solani was prepared in 500 ml Erlenmeyer flasks containing potato-sugar infusion (IPA). The flasks were sterilized in autoclave at 120 °C for 15 min. Five cylinders 5 mm in diameter containing the antagonist or R. solani were aseptically transferred to the flasks and incubated for 10 days in a Lab-Line^® shaking incubator at 110 revolutions per minute (rpm); mycelium was removed from the flasks and grinded in a blender.

From Petri dishes containing F. oxysporum or P. capsici mycelium, cylinders 5 mm in diameter were taken and sown in IPAA and in V8 juice agar (V₈A), respectively; then, they were incubated in darkness at 27±1 °C. Seven days later, 10 ml of 0.9 % (Pisa^®) sodium chloride isotonic solution were added to the Petri dishes containing P. capsici. Later, the solution was removed, and, using a dissection needle, the medium was divided in six fragments. Each fragment was transferred to a Petri dish containing 10 ml of sterile running water.

The dishes were kept under light for 48 h at 26 °C and 48 h in darkness at 28 h °C. After this period, they were placed at 4 °C for 20 min and then at 27±1 °C for 1 h to induce zoospore release. The number of antagonist and pathogen propagules was counted in a hemocytometer and adjusted to the inoculum density at 1X10⁸ for the antagonist, and at 1X10⁷ ufc for F. oxysporum and P. capsici; for R. solani, 1 g of previously grinded mycelium was added. The antagonist inoculation was made in seeds, when the seedlings were transferred to trays, plus two 10 ml applications three days later, one day before transferring the plants to pots, and immediately after transplanting them to pots. The patoghen inoculation was made three days after the antagonist was inoculated. All of them were applied at the base of the stem.

Evaluated variables

Records were taken of the incidence (%) of plants showing disease symptoms and severity (damage level), plant height, diameter of the base of the stem, as well as root and canopy dry weight. Observations were made every day to record the first two variables. P. capsici severity was evaluated using the scale proposed by ^{Morán-Bañuelos et al. (2010)}; F. oxysporum, using the scale proposed by ^{Marlatt et al. (1996)}, and R. solani severity, using the scale proposed by ^{Navarrete-Maya et al. (2009)}. When the experiment ended (3 months after transplanting), the height of the plants and the diameter of the base of the stem were measured. Roots were cut at the base of the stem. Both canopy and roots were placed in Kraft paper and dried at 60° C on a stove (Felisa®) during 4 days. Five plants from each treatment were randomly taken to record their dry weight.

Statistical analysis: Data from the evaluated variables in all the trials were used to perform normal distribution (Shapiro-Wilk and Kolmogorov-Smirnov) and variance homogeneity (Bartlett) tests. For data obtained from the greenhouse trial we had to use a logarithmic transformation. Variance analysis (ANOVA) and means comparison (Tukey, P≤0.05) were performed using the SAS version 9.0 software. The analysis was performed with data from the two experiments.

Mycelial interaction between Saccharicola sp-P. capsici and Saccharicola sp-R. solani.

Under in vitro conditions, interaction between Saccharicola sp and P. capsici was observed 24 h after mycelia of both microorganisms came into contact, and the attachment phenomenon was observed (Figure 4). At 48 h, formation of Saccharicola sp pseudoparenchymal cells was observed on P. capsici hyphae, and at 72 h, growth around P. capsici hyphae (winding) (Figure 4). At 96 h, formation of P. capsici sporangia was observed, but in the controls sporangia appeared until day 5. At day 7, P. capsici hyphae showed vacuoles, cytoplasm granulation, deformation, and appeared to be empty (Figure 4).

Figure 4. Mycelial interaction between Saccharicola sp-P. capsici. A) Saccharicola sp winding around Pc hyphae 72 h after mycelia of both organisms came into contact (hpem), B) hyphae showing vacuoles (v) and cytoplasm granulation, C) deformed hyphae and seeming to be empty 7 days after mycelium of both microorganisms came into contact, D and E) P. capsici control at 72 h and 7 days after, respectively. 

In R. solani, starting 24 h after mycelia of both microoganisms came into contact, sinking was observed in R. solani hyphae at the points of contact with Saccharicola sp (Figure 5); at those points we also observed the formation of small structures on Saccharicola sp. hyphae. At 96 h, at the points of contact with Saccharicola sp, R. solani hyphae showed deformation, and the mycelium, less intense coloration (Figure 5). At the points of interaction between the antagonist hyphae and the pathogens, we observed that the antagonist hyphae were smaller in diameter, which suggests that young mycelium is the one that comes into contact with the pathogen hyphae.

Figure 5. Mycelial interaction between Saccharicola sp-R. solani (A-Rs). A) Rs hyphae (h) sinking at the points of contact with Saccharicola sp mycelium 24 h after the mycelium of both organisms came into contact (hpem). B) Rs hypha deformed and with less intense coloration at 96 hpem. 

Scanning electron microscopy (MEB)

In the interaction Saccharicola sp-P. capsici, winding of the antagonist hyphae around P. capsici mycelium was observed (Figura 6), as well as formation of anchored structures on the same phytopathogen.

Figure 6. Scanning electron microscopy. A) Saccharicola sp winding around Pc hyphae; B) Saccharicola sp anchoring structure on Pc. 

Saccharicola sp antagonist activity in the greenhouse

Under greenhouse conditions, P. capsici and F. oxysporum were pathogenic to chili pepper. The first caused 100 % disease incidence and 50 % mortality in infected plants. In F. oxysporum, 75 % incidence was recorded and 50 % dead plants; R. solani was not pathogenic to chili pepper. When the antagonist was inoculated along with the three pathogens, both in sterile soil and naturally infested soil, there was 100 % incidence in the chili pepper plants. The antagonist had no favorable effect on the diameter of the base of the stem, height or canopy and root dry weight. It was obvious that the application of Saccharicola sp did not protect the chili pepper plants from phythogens attack nor improved the plant development.