Cocultivation of Pleurotus ostreatus (Jacq.) P. Kumm. with yeasts

Cocultivation of Pleurotus ostreatus with eight yeast species were investigated on water agar. Special mycelial structures contacting with yeast cells were found in such cultures: nipple-like appendages and coralioid hyphae. Three out of eight species, Hanseniaspora uvarum, Rhodotorula minuta, and Saccharomyces cerevisiae were identified as trophic preferendum for P. ostreatus. These three yeast species were used for mushroom cultivation on sunflower seed peel. The biomass of fruiting bodies increased by 52.8–75.7% with the H. uvarum and S. cerevisiae suspension presence in the substrate.


INTRODUCTION
Oyster mushroom, Pleurotus ostreatus (Jacq.) P. Kumm., is one of the most widely distributed mush room of Pleurotus genus intensively cultivated in vari ous countries of the world. In mushroom culturing, the oyster mushroom is appreciated due to its gusta tory properties and expressed "mushroom" odor as well as its indiscriminateness to conditions of cultiva tion at various substrates of plant origin [1].
The oyster mushroom belongs to the ecological trophic group of xylotrophic mushrooms. For all tree destroying mushrooms, the main growth limiting fac tor is nitrogen since C : N ratio in wood substance var ies from 300 : 1 to 1000 : 1 and even more, while 30 : 1 ratio is optimal for growth of a majority of mushrooms [2]. For many xylotrophic mushrooms, an ability to compensate nitrogen deficiency owing to parasitism on bacteria, yeasts or algae, persisting on wood sub strate, was declared [3,4]. Epiphytic yeasts are widely distributed in nature at surfaces of footstalks, leaves, fruits and berries of grassy and woody plants as well as at the cortex of trees. Yeasts do not directly participate in the process of wood destruction since they do not have the necessary enzymes; however, yeasts serve as nitrogen source for xylotrophic yeasts thereby influ encing processes of wood destruction [5].
The feature of the oyster mushroom to use micro organisms as nutrient source may be successfully used in biotechnology and mushroom culturing for increas ing of eatable mycelium biomass: for example, cocul tivation of the oyster mushroom with Azospirillum brasilense bacteria in the liquid medium allows us to increase the output of mycelium biomass by 30% and to reduce the time for culturing of the growth myce lium [6]. To stimulate growth and fruiting of mush rooms, from the viewpoint of safety for health of con sumers, it is preferable to use biological objects (bacte ria, yeasts) nonpathogenic for humans instead of the addition of chemical compounds-regulators. More over, addition of microorganisms to substrate of live culture allows mushroom mycelium to use it as nutri tion during the whole period of cultivation.
The aim of the present work is to investigate inter actions between oyster mushroom and yeasts in the process of vegetative growth of mycelium in culture and the influence of yeasts on the process of mush room fruiting. created via addition of water suspension of yeast (con centration 10 5 -10 7 cells per ml) growth on slant wort agar (2.2%) through dropping (50 µl) at a distance of 1.5-2 cm from the edge of mushroom colony by the seventh day of mycelium growth. Microscope investi gation was performed by the third to fourth day of coc ulturing using Axioskop 40 FL device with AxioCam MRc. All experiments were done in triplicate.

Objects of investigation. Xylotrophic basidiomycete
Obtaining of fruiting bodies. Fruiting bodies of the oyster mushroom were obtained on sunflower seed peel in conditions of climatic camera. Seed mycelium was grown on wheat grains with 3% chalk stone for 14 days in the thermostat at 25 ± 1°C. Cultivation sub strate was prepared according to a modified technique of Stamets [7]. Humidity (moisture content) was detected using a well known method: humidity (%) = (mass of wet substrate -mass of dried substrate) / mass of wet substrate ×100% [8]. Forcing of fruiting bodies was performed at a temperature of 20 ± 1°C and air relative humidity of 95%. After inoculation of sub strate with mycelium of the oyster mushroom, water suspension of yeasts (concentration 10 5 -10 7 cells per ml), 10 ml per each flask, was added to substrate. Two control variants were used: substrate without any sup plements (control no. 1) and substrate with addition of sterile water (10 ml per each flask) (control no. 2). All experiments were performed in triplicate.

RESULTS AND DISCUSSION
Cultivation on agar medium. Mycelium of the oyster mushroom was totally overgrown with microcolonies of yeasts at the surface of water agar. In cocultures with C. albidus, H. uvarum, M. pulcherrima, and S. cerevi siae, there is 4-8 fold increase of frequency of branching of mycelium hyphae. Significantly branch ing mycelium hyphae form coralioid structures [3]; these structures were observed in cocultures with H. uvarum, R. minuta, and S. cerevisiae. In cocultures with C. albidus, D. hansenii, K. marxianus, and S. cer evisiae, there were mycelial rings (Fig. 1a); it presents a part of twirly twisted vegetative hypha and phenotyp ically similar to chasseur loops that form mycelium of ravenous fungi as well as to P. ostreatus in the presence of nematodes in culture [2]. Functions of mycelial rings of the oyster mushroom are not fully clear: prob ably, they are formed not for binding of yeast cells but act as physiological response of mycelium to the pres ence of yeasts in the culture.
Both in the cases of monoculture of the oyster mushroom and cocultures with all yeasts species, except for R. minuta, there were capitate outgrowths (Fig. 1b). In the apical part of the outgrowth, there are drops of secrete. This secrete is active against nema todes: these outgrowths are formed in the oyster mushroom and in ravenous fungi and serve for physi cal inactivity of nematodes [2]. However, functions of secrete produced by capitate outgrowths of the oyster mushroom are not totally known. Probably, mycelial cells produce not only toxin for nematodes but also some other compound. In all cocultures, formation of special nipple like short appendages that connect mycelium and yeast cells was detected (Fig. 1c, 1d) [9]. Nipple like appendages were rarely seen in cocultures with C. albi dus, C. capitatum, D. hansenii, K. marxianus, S. cerevi siae 3785; from one to three in the visual field are in cocultures of M. pulcherrima, R. minuta, S. cerevisiae 3809, S. cerevisiae Moment. It formed especially intensively in coculture of the oyster mushroom with yeasts H. uvarum (more than three in one visual field). Contacts between hyphae with yeast cells were detected in cocultures with C. albidus, C. capitatum, H. uvarum, R. minuta, M. pulcherrima, S. cerevisiae 3785, 3809 and Moment, especially intensively (more than three in one visual field) with H. uvarum. Con tacts were formed only in those cocultures where sus pension of live yeast cells was introduced. After intro duction of boiled cells, contacts with yeast cells and nipple like appendages were not formed.
Trophic preferendum for P. ostreatus was detected on basis of the following features: 4 fold increase in the frequency of hyphae branching, intensive forma tion of nipple like appendages (two or more in visual field), coralloid structures, and numerous contacts of mycelium with yeast cells (two or more in visual field). Thus, three of eight yeast species, H. uvarum, R. minuta and S. cerevisiae, were chosen as trophic preferendum. These yeast species were used in the process of cultivation of the oyster mushroom on sun flower seed peel.
Obtaining of fruiting bodies. In all samples supple mented with yeasts (H. uvarum, R. minuta, S. cerevi siae), there was a total overgrowth of a substrate with mycelium by the 14th day (in control nos. 1 and 2, it was by the 18th day); thus, forcing of fruiting bodies in samples with yeasts was detected 4 days earlier. Incu bation duration (time since start of forcing to period of collection of fruiting bodies) was almost the same while culturing of the oyster mushroom without and with yeasts: fruiting bodies were cut off by the 11th-13th day in all samples.
Humidity of the substrate after addition of sterile water and suspension of yeast cells was changed mini mally inside of a norm stated for cultivation of the oys ter mushroom [10]. In control no. 1, substrate humid ity was 61.2 ± 2.0%; in control no. 2 and in experimen tal samples, it was 60.5 ± 1.5%. Thus, influence of substrate humidity on the rate of its overgrowth with mycelium may be ignored: increase in mycelium growth is due to addition of yeast cells to suspension substrate.
Fruiting bodies of P. ostreatus were phenotypically similar at cultivation with yeasts or without them. Mushrooms were formed individually or in groups on 2-3 fruiting bodies. It was noted that, in control nos. 1 and 2 and in the presence of R. minuta, yeasts fruiting bodies are higher (to 75 mm) and thin, while in the presence of H. uvarum and S. cerevisiae they are short (to 60 mm) and more thick. Significant differences in biomass in control nos. 1 and 2 and in the presence of R. minuta yeasts were not detected. Biomass of fruiting bodies was higher than in control: by 75.7% in the presence of H. uvarum and by 52.8% in the presence of S. cerevisiae (table).
Maximal increase in biomass of harvest was detected in the presence of H. uvarum yeasts, which correlates with maximal amount of nipple like appendages and contacts between mycelium and H. uvarum yeast cells on agar medium. Viable yeast cells were found in the substrate by the 14th and 28th days of cultivation during the whole period of mycelial growth and fruiting. Probably, the oyster mushroom uses yeast cells as additional nutrient source during its growth at the substrate.
The obtained data demonstrate the ability of the oyster mushroom, P. ostreatus, transform to parasitism and to use yeast cells as nutrient source in laboratory conditions on water agar, i.e., on medium without sources of carbon and nitrogen needed for the mush room. Furthermore, different level of parasitic activity toward various species of mushrooms is observed. The introduction of suspension of live yeast cells results in increase of growth rate for the mushroom at the sub strate and increase in biomass of fruiting bodies.