Materials and Methods
- New strains obtained after UV treatment and protoplast fusion of native Trichoderma harzianum: their biocontrol activity on Pyrenochaeta lycopersici
UV-A and UV-C irradiation. Th11, Th12 and Th291, native T. harzianum strains, previously selected from suppressive soils where tomatoes were grown for more than 15 years without the use of MeBr, that show a good behavior as BCAs of P. lycopersici (Pérez et al. 2002; Besoain et al. 2003), were used. This isolates were kept lyophilized and stored at -20ºC, for a five-year period. These were recovered in Potato Dextrose Agar (PDA) plates, and conidia were obtained as described (Pérez et al. 2002). Later, 100 µl of suspensions of each strain, containing 1 x 103 cfu/mL, were spread on plates containing corky root medium (CRM) (Shishkoff, 1992), and irradiated with 0.01-0.1 J/cm2 doses of UV-C light (254 nm) or with UV-A light (320 nm), during different time periods (10, 20, 40, 80 and 160 min). After irradiation, plates were incubated at 23ºC for 7-10 days. Grown colonies were purified, lyophilized and stored at -20ºC for further tests.
Protoplast fusion. New strains from Th11, Th12, ThV and Th291 native T. harzianum were obtained, using a combination of the Bae and Knudsen (2000), the Stasz et al. (1988) and the Sivan et al. (1990) techniques. Briefly, 3 x 105 conidia of each native strain were seeded in flasks containing 100 mL of sterile yeast extract broth. Th11, Th12, ThV and Th291 were combined in order to obtain six different series: ThF1 (Th11 x Th291), ThF2 (ThV x Th291), ThF3 (ThV x Th11), ThF4 (ThV x Th12), ThF5 (Th11 x Th12), and ThF6 (Th12 x Th291). Flasks were incubated for 23 hrs at 28ºC in constant agitation. The mycelium obtained was collected and incorporated to 10 mL of a cell wall lytic enzymatic solution (L3768, Sigma), and incubated again with agitation during four hrs at the same temperature. Finally, the suspension was filtered through four layers of cheesecloth and centrifuged at 100 x g during 10 min to collect protoplasts or spheroplasts. These were washed in STC buffer (1.2 M sorbitol, 10 mM Tris-HCl pH 7.0, 10 mM CaCl2, Autoclave) to remove the enzyme solution, centrifuged again at the same speed and time, suspended in 1 mL of STC buffer. Protoplasts or spheroplasts were counted in a Bauer hemacytometer. 200 µl of a solution containing 60% (w/v) polyethyleneglycol (PEG), 10 mM CaCl2 and 10 mM Tris-HCl pH 7.5, were gradually added to the protoplast suspension (1 x 105 protoplasts/mL of STC), incubated for a few min and then, a second and a third aliquot of 500 µL of the same PEG solution were added. The mixture was incubated for 10 min at 30ºC, with agitation, to induce fusion. An extra volume of STC (2.2 mL) was added before centrifuging at 100 x g. Protoplasts were diluted in STC to obtain serial dilutions. Aliquots of 100 µL were spread on plates containing protoplast regeneration medium (PRM), (Stasz et al. 1988). Plates were incubated at 23ºC during a week for the obtainment and purification of strains.
The preliminary selection of new strains was done on the basis of their growth at low temperatures and high pH, and on the innocuousness on tomato plant growth. The additional selection took into account % inhibition of P. lycopersici growth in dual cultures and due to volatile and/or diffusible metabolites production.
Growth at different temperatures and pH. The selection of new strains obtained both after UV light irradiation or protoplast fusion, as well as the behavior of their corresponding native isolates, was carried out by seeding a mycelium plug on PDA plates and incubated at 10ºC and 15ºC for seven days. Growth at different pH values was estimated using PDA plates adjusted to pH 6.0, 6.5, 7.0, 7.5 and 8.0, after autoclaving, with KOH or HCl. Plates were incubated at 20ºC for seven days. Each experiment was run in triplicates. Results were analyzed using the Tuckey test at 5% significance.
Innocuousness tests of native and new strains on tomato plants. (a) New strains obtained after UV irradiation. A sterile distilled water suspension (5 x 105 conidia/mL) of each native or new strain of T. harzianum obtained after UV irradiation was prepared, to be used as inocula for the evaluation of possible damages or benefits of these strains on tomato plants. Tests were run as follows: tomato fine roots were submerged in the conidia suspension, and once inoculated, seedlings were transplanted to pots containing a mixture of compost:sand:soil = 3:1:1 (v/v/v), and kept under controlled greenhouse conditions (controlled range of temperature: 12ºC (minimum) and 20ºC (maximum); 12 hrs natural light, irrigation: once a week). One month after transplantation, they were pulled out for evaluation. Each unit of treatment considered five plants of each of the four cultivars used (Fortaleza, R593, Naomi and Yonit), per each of the strains tested. Pots were arranged in a complete random design. Once the plants were removed, the potting mix was rinsed from the roots and the total fresh weight was measured. Then, plants were dried in paper bags for 48 hrs at 60ºC to obtain the total dry weight. Treatments considered a single new strain. Controls were run with the parental strains. Results correspond to mean and were analyzed by the Tuckey's test at 5% significance.
(b) New strains obtained through protoplast fusion. Pellets that contained the different strains of Trichoderma were prepared according to Montealegre and Larenas (1997) and used as inoculums for the evaluation of possible damages or benefits on tomato plants varieties Yonit and Dalila (Zeraim Gedera) cultivated in a cold greenhouse in Quillota, V Region, Chile. Nine plants were used in each treatment, with a completely randomized design. Treatments considered the use of native isolates, Th291, Th11, Th12 and Th650, the new strains obtained through protoplast fusion F1-2, F4-4 and F5-8 the UV-C irradiated new strain Th11C80.3, and a commercial product based on T. harzianum (Orious, powder formulation). Controls were run without the addition of BCAs. Seedlings were transplanted into plastic bags containing natural infested clay soil with P. lycopersici, obtained from a monoculture tomato crop in the Quillota area. Recently prepared pellets containing new and native strains, treatments were used in 1-3 g doses, depending on the concentration of each BCA, previously determined by viability tests. Pellets were set in the planting hole before transplanting. Two months later after transplanting, the plants were pulled out for evaluation. Once the plants were removed, the potting mix was rinsed from the roots and the total fresh weight was measured. Then, plants were dried in paper bags for 48 hrs at 60ºC to obtain the total dry weight. Treatments considered a single new strain. Controls were run with the parental strains. Results correspond to mean and were analyzed by the Tuckey's test at 5% significance.
Dual cultures. The native isolates of T. harzianum, as well as the new strains obtained either after UV light irradiation or protoplast fusion, were spread on acidified PDA plates (1mL of 1M lactic acid per L of PDA medium), placing two equidistant 0.5 cm diameter agar disks, one containing young mycelium of BCA (Tewari and Bhanu, 2004) and the other, P. lycopersici mycelium. Controls were run placing two equidistant 0.5 cm agar disks of the BCA or the phytopathogen. Plates were incubated for 7 days at 23ºC. The area of the pathogen colony was measured and compared to the control. Each experiment was run in triplicates. The pathogen growth inhibition was calculated as follows:
Pathogen growth inhibition (%) = 100 - % Control
Control (%) = ACP/ACC x 100; (ACP: colony area of pathogen with biocontroler; ACC: colony area of pathogen with same pathogen)
Production of diffusible metabolites. PDA containing plates were inoculated with a 5 mm disk of each of the native and the new strains of Trichoderma. The inoculum was placed on a cellophane paper disk (non water proof, 0.6 µm pore size as in Kosuta et al. (2003)) which covered completely the plate, as described by Whipps (1987). After incubation at 15ºC and before the mycelial growth reached the edges of the cellophane paper, it was removed along with the grown Trichoderma. The plate was inoculated again at the centre, but this time with a 5 mm disk of pure mycelia of P. lycopersici. The plates were further incubated at 15ºC for seven days and the pathogen growth was measured. Mocked inoculated plates with a 5 mm disk of PDA and inoculated with P. lycopersici on the cellophane paper as above, were used as controls. The experiment, considering a single Trichoderma strain, was run in triplicates. Results were analyzed using the Tuckey test at 5% significance.
Production of volatile metabolites. In order to evaluate the presence of volatile metabolite in mutants or native isolates of T. harzianum, the methodology described by Whipps (1987) was used. Two PDA containing Petri dishes (half plates of the same diameter) were set against each other after inoculation, and were sealed with waterproof adhesive tape, to avoid leakage of metabolites. Inoculation was done as follows: one half dish was inoculated with a 0.5 cm diameter disk of young mycelium of the BCA, and the other half dish was inoculated with P. lycopersici, this last half dish was placed on top. Due to the slow growth of P. lycopersici, it was seeded three days before Trichoderma strains.The plates were incubated at 15ºC for four days and the growth of the pathogen was measured and compared to controls run using the same inoculum in both half dishes. The experiment, considering a single Trichoderma strain, was run in triplicate. Results were analyzed using the Tuckey test at 5% significance.
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