P. albidus: Argentina, Buenos Aires, La Lucila, in canker of Salix humboldtiana, 5-V-1996, BAFC 2787; La Plata, on stump, III-1996, coll. J. Deschamps, BAFC 809; Pergamino, on Salix sp., 6-IV-1996, coll. E. Albertó, BAFC 695; Tigre, on Populus sp., 10-III-1997, coll. Claudio Lázzari, BAFC 136; Córdoba, La Punilla, on Populus sp., 25-II-2001, coll. N. Manero and B. J. Lechner, BAFC 190. P. cystidiosus: Argentina, Capital Federal, Barrio La Paternal, on dead zone of Platanus sp., coll. S. Frachia, 18-IV-2000, BAFC 188; Buenos Aires, La Plata, Plaza San Martín, XII-27-1994, leg. H. Spinedi, BAFC 73. P. djamor var. djamor: Argentina, Misiones, El Palmital del cruce, 28-V-2001, coll. E. Albertó, BAFC 821. P. djamor var. roseus: Argentina, Misiones, El Palmital del cruce, 28-V-2001, coll. E. Albertó, BAFC 815. P. ostreatus: Argentina, Capital Federal, 18-IV-1994, coll. Pablo Pica, BAFC 2034; Neuquen, Moquehue, on trunk of Araucaria araucana, III-1993, coll. J. del Vas, BAFC 120; Italy, commercial strain, IX-1993, BAFC 2067. P. pulmonarius: Buenos Aires, Ezeiza, on living declining tree of Populus sp., 25-VII-1987, coll. J. Deschamps, BAFC 1003; Misiones, San Pedro, on branches of Araucaria angustifolia, 27-V-2001, coll. E. Albertó and O. Popoff, BAFC 76; San Pedro, S 26° 32′, W 54° 04′, on branches of A. angustifolia, 27-V-2001, coll. D. Krueger, BAFC 213; same location, same host, same date, coll. E. Albertó, BAFC 263.

Cultures were inoculated with 7mm diameter cylinder in 90mm Petri dishes containing Nobles’ medium16 and incubated in the dark at 5, 12, 20, 25, 30 and 35°C. Growth of mycelium was measured as radio of the colony in duplicate with a ruler (0.5mm scale).

It was prepared in 750ml glass bottles filled with boiled wheat grains (Triticum durum) and 1% w/w calcium carbonate (CaCO3). Bottles were sterilized for 1.5 hour at 121°C, cooled and then inoculated with 1cm diam plug of mycelium grown on Nobles’ medium. Bottles were incubated at 25°C, in the dark, with periodical shaking. Time required for spawn production was recorded.

To obtain fruiting bodies, standard methods for fruiting species of Pleurotus were used22,28. Three substrates were used based on sawdust of Salix sp. and wheat straw, supplemented with wheat meal and oatmeal (Table 1). Three hundred grams dry weight of each mixture was introduced into polypropylene bags of 20×40cm for sawdust and 30×45cm for wheat straw. Final humidity in the substrate was adjusted (w/w) to 74% accounting for the initial humidity content of substrate. Bags were stopped with cotton plugs held by PVC (polyvinyl chloride) cylinders and autoclaved at 123°C, 1.2 Kg/cm2, for 2h. After cooling, the bags were inoculated with 3% (wet weight) of spawn and incubated at 25°C in the dark until the complete colonization of the substrate.

Six small cuts (20mm long) were regularly made on the bag surface. Bags were then moved to a culture room (2.5×4.5 m) for fruiting bodies production; they were kept at 18-20°C with 9h light/15h dark photoperiod (20W fluorescent light), 75 to 85% humidity levels, and watering by spray (fog type) for 5min every 3h which was automatically provided.

Three to four flushes were collected during the cropping period (time lapsed between the induction day and the last harvest day) defined in 120 days. Mature fruiting bodies were collected and the following production and morphological traits were registered: A) Production: i) Primordia initiation (in days) from the start of incubation; ii) BE: fresh fruiting bodies weight (total yield)/dry substrate weight (expressed as a percentage). B) Morphological traits: i) pileus breath and length; ii) stem length. After four months under fruiting conditions, bags were dried and weighed; dry matter content loss was calculated as a percentage.

Six bags were used for treatment. Tukey HSD test was used to determine significant differences between groups in an ANOVA. The normality and homogeneity assumptions were checked by means of KS and Bartlett tests respectively for the validity of ANOVA method.

The effect of temperature on mycelium growth is shown in Fig. 1. Differences in the rate of growth can be observed either interespecifically or intraspecifically. The highest growth rate recorded was reached by strains BAFC 263 of P. pulmonarius with 7.4mm/day which had significant differences among strains of this species (p<0.01), BAFC 190 of P. albidus, with 6.16mm/day which had significant differences among strains of this species (p<0.01), and BAFC 2034 of P. ostreatus with 5.8mm/day which had only significant differences with strain BAFC 2067. The lowest growth rate was shown by P. cystidiosus, followed by P. djamor. For the former, BAFC 188 and 73 reached hardly 1.2 and 0.5mm/day respectively, while BAFC 821 and 815 of P. djamor reached 1.5 and 0.5mm/day. Among strains of P. ostreatus, P. albidus and P. pulmonarius no meaningful differences were observed. P. albidus, P. djamor, P. ostreatus and P. pulmonarius reached their optimum growth at 25°C, except strains BAFC 136 of P. albidus and BAFC 76 of P. pulmonarius which reached it at 30°C. The optimum temperature of growth for P. cystidiosus was 30°C. Strains BAFC 136, 215, 695, 809, 2545, 2787 of P. albidus grew 29 to 50% less than strain BAFC 190. No meaningful differences were obtained between BAFC 2034 and 120 of P. ostreatus, while BAFC 2067 grew 14% less than BAFC 2034. Strain BAFC 263 of P. pulmonarius grew 28% more than BAFC 76 and 1003.

Figure 1.

Effect of temperature on mycelium growth of naturally occurring Pleurotus species from Argentina. A) P. albidus. B) P. cystidiosus. C) P. djamor. D) P. ostreatus. E) P. pulmonarius.

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Strains of P. albidus, P. ostreatus and P. pulmonarius, which had highest growth rate on Nobles’ medium, required shorter time for spawn production: 8 to 12, 8 to 10 and 5 to 10 days respectively. Strains of P. djamor and P. cystidiosus were slower and required 11 to 12 days and 15 to 20 days respectively to completely colonize grains.

The range of time required for incubation (time of colonization) on different substrates at 25°C also depended on species and varied from 15 to 45 days. Strains of P. albidus, P. ostreatus, P. djamor and P. pulmonarius required 15 to 20 days, meanwhile strains of P. cystidiosus required from 28 to 45 days (data not shown).

P. albidus required 20 to 42 days after spawning for primordia development, P. djamor, 27 to 37 days, P. ostreatus 28 to 42 days, and P. pulmonarius 25 to 40 days (Table 2). P. cystidiosus needed more than 45 days (data not shown).

The effect of different substrates on fruiting bodies production of naturally occurring strains was determined (Table 2). The most suitable combinations of substrate and strain were obtained with P. albidus BAFC 2787 on SW, and BAFC 190 on W, which reached a BE of 171.3 and 161.7% respectively, being the highest yields obtained in this study. Regarding the other strains of P. albidus, the statistical analysis showed that there were significant differences (p≤0.05) among strains BAFC 190, 809 and 695 but no significant differences (p > 0.05) were found among substrates. When the morphology of fruiting bodies obtained with P. albidus was studied (Table 3), we observed that no significant differences were found between width and length of pileus. Strain BAFC 2787 produced the largest stems on SW. Only significant differences (p≤0.05) for stems length were found between BAFC 2787 on SW and BAFC 136 on SS.

BEs values of P. djamor strains varied from 61 to 112% (Table 2). Strains BAFC 815 on W, and 821 on SS and W, reached the highest BE with significant differences compared with the other treatments for the same strains. The widest pileus was obtained with strain BAFC 815 on W showing only significant differences with SS for both strains. When the length of pileus was compared, we found that it was minor for BAFC 821 on SS but only significant differences were obtained between SS and SW. No significant differences were observed when stem length was compared (Table 3).

The best yield of P. ostreatus was produced by BAFC 2034 on SW reaching BE 122.9 (Table 2). Similar values were obtained with BAFC 2434 on W and 2067 on SW. The analysis of BEs of P. ostreatus strains showed that BAFC 120, which developed several aborted primordia, produced lowest yields (BE 37 to 61.7). For this species, SS produced the lowest BE, showing significant differences with W and SW. The study of morphology showed that largest sizes of pileus were obtained with the strain BAFC 120 on SW. The highest stem size was produced by strain BAFC 120 on SW and showed significant differences (p≤0.05) with strain BAFC 2034 on SS and BAFC 2067 on all substrates (Table 3).

Yields obtained with strains of P. pulmonarius were very variable. The best yield of this species was produced by strains BAFC 76 and 1003 (BE 97.9 to 128%) showing significant differences (p≤0.05) with BAFC 263 which produced lowest yield in this species (Table 2). The morphological study showed that no significant differences in the size of the pileus were found among strains when cultivated in different substrates. Stems obtained with strain BAFC 76 on SS were significantly shorter than those obtained with BAFC 1003 on W.

Yields of P. cystidiosus were very poor with BE values of 0-30%; these were the lowest reached in this study (data not shown). SW did not produce fruiting bodies. Due to the low yields obtained with this species, morphology traits where not studied.

Dry matter loss for P. albidus varied from 53.3±4.7 on SS for strain BAFC 695 to 85.3±6.8 on SS for BAFC 2787 with significant differences (p<0.05). P. djamor did not show significant differences in dry matter loss varying from 72.7±1.2 on W to 79.7±9.8 on SS for BAFC 821. P. ostreatus varied from 69.2±2.6 for BAFC 120 to 86.8±3.3 for BAFC 2007 both growing on SW with significant differences (p<0.05). For P. pulmonarius the loss varied from 68.1±0.8 on SW to 85.2±1.9 on SS both for strain 1003 with significant differences (p<0.05). Dry matter loss of the substrates was generally in agreement with BE increase (Table 2). Nevertheless, several strains with low BE, such as BAFC 120 (on all substrates), 2067 (on SS) of P. ostreatus and BAFC 263 (on SW) of P. pulmonarius, showed high dry matter loss (Table 2). Also strains with high yields had comparatively lower substrate dry matter lost as BAFC 809 on SS or 2787 on SW.