BIOLOGIA PLANTARUM (PRAHA)

16 (6) : 436--443, 1974

Stimulation of the Formation of Fruiting Bodies of the Fungus Lentinus tigrinus (BULL.) Ft. by Growth Regulators

Z. SLADKY and V. TIOH:~

Department of Plant Biology, Faculty of Natural Sciences, J. E. Purkyni~ University, Brno

Received October 29, 1973

Abstraet. Growth regulators, indole-3-aeetie acid (IAA), gibbereUic acid (GA3), and kinetin (KIN), were used in different concentrations to stimulate the initiation and further development of the fruiting bodies of the fungus Lentinus tigrinus. Vegetative myeelium of the fungus was cultivated on cellulose cylinders soaked with a synthetic nutrient solution or with a 3% malt extract. When the mycelium covered the surface of the cylinders, further cultivation was carried out in graduated concentrations of the growth regulators mentioned above. The number of developed fruiting bodies showed that the optimum IAA and GAa concentrations were in both media 300 p.p.m. The optimum concentration of kinetin was 400 p.p.m.

The individual growth regulators influenced characteristically also the shgpe of the fruiting bodies and changed in them the natural level of endogenous growth regulators. The addition of IAA into the medium raised the level of endogenous auxins in the cap. The presence of gibberellic acid and of kinetin in the medium resulted in an increase in the level of these regulators in the fruiting bodies.

The regula t ion of g rowth and deve lopment of f rui t ing bodies of mos t fungi is still a difficult problem. I t can be supposed t h a t some regu la to ry role is p layed in these processes by g rowth regulators. I t was es tabl ished in previous exper iments (RYPAGEK and SLADKY 1972, 1973) t h a t some sig- nif icant stages in the course of deve lopment of the fungus Lent inus tigrinus are accompan ied b y character is t ic changes in the ra t io be tween endogenous g rowth regulators . E a r l y stages of the vege ta t ive deve lopmen t of the my- cel ium are charac ter ized b y a re la t ive ly high level of gibberellins and ey to- kinins. On the con t ra ry , the ini t ia t ion of the pr imordia of f ru i t ing bodies is accompan ied by a decrease in the con ten t of g rowth s t imula tors and b y the occurrence of inhibitions. These inhibi t ions are considered to be one of the causes of the s low-down in the fo rma t ion of f rui t ing bodies f rom the pr imord ia (PARK 1963). URAYAMA (1961) assumes t h a t fungal inhibi tors can be decomposed by cer ta in specific bac ter ia and he uses these bac ter ia on the basis of his hypothes is to enhance the fo rma t ion of f rui t ing bodies in the cul tures of the fungus Agaricus bisporus.

Address: 611 37 Brno, Kotls163 2, Czechoslovakia.

436

FORMATION OF FRUITING BODIES OF L E N T I N U S 437

The aim of our paper was to overcome the inhibitions which retard the growth and the development of the fruiting bodies of the fungus Lentinus tigrinus by exogenously supplying synthetic growth regulators to the cul- tivation medium. The results obtained in earlier experiments (RYPA6EK and SLADK~ 1972) enabled us to choose suitable synthetic growth regulators and to asses the range of useful concentrations. The total production of the fruiting bodies and the character of the endogenous growth regulators in them served as criteria for the evaluation of the effect of the growth regulators tested.

Material and Methods

The fungus Lentinus tigrinus (BULL.) FR., strain Nr. 30, from the collection of pure cultures of the Department of Plant Biology were used in the experiments. The myeclium was inoculated under aseptic conditions on cellulose cylinders 3 cm in diameter and 5 cm high. This substrate was soaked in the first experiment with the nutr ient solution of this composition:

K2HPO4 1.0 g vitamin BI 1.0 mg MgS04.7H20 1.0 g vitamin B6 1.0 mg AZ solution 1.0 ml vitamin H 1.0 mg glucose 20.0 g distilled water 1 000 ml asparagine 2.0 g

In the second series of experiments cellulose cylinders were soaked with a 3% malt solution. The mycelium covered in both experiments the entire surface of the cellulose substrate in

3 weeks in which time the primordia of the fruiting bodies appeared sporadically as well. The cylinders were then transferred to crystallization dishes containing 80 ml of growth regulator solutions of different concentrations. The concentration range was broadened in the second series of experiments to: 25, 50, 100, 300, and 400 p.p.m, of indole-3-acetic acid (IAA), gibberellie acid (GA3), and kinetin (KIN) respectively. Further cultivation was performed under non- aseptic conditions, at room temperature, at high relative air humidity, and under daylight diffuse illumination. Regular ventilation was secured in all the combinations.

Fruiting bodies developed during the next 20 days on the cylinders, the size, number, and fresh weight of which was followed. The values characterizing the development of the fruiting bodies on the synthetic medium are given in Table 1 and the values obtained in the second experiment with 3 % malt solution are presented in Table 2. The fruiting bodies from the optimum concentrations in the second experiment were used for analytical determination of the character of endogenous growth regulators similarly as in previous studies (RYPX~EK and SLADK~2 1972).

For the extraction of the endogenous growth regulators 10 g f.w. stalk or cap samples were taken from the cultures with the optimum concentrations of individual synthetic growth regu- lators. After separating the extract, auxins were determined using the averts coleoptfle bioassay after BENTLEY and tIousL~.Y (1954). Gibberellin-like substances were determined by means of the lettuee-hypoeotyl bioassay described by FRANKLAlVD and WAREnVG (1961), and eytokinins were followed on barley leaf segments according to the bioassay of OSBORNE and McCALLA (1961). The values obtained are presented in the histograms in Fig. 1. A quantitat ive comparison was made possible by denoting the effects of 1 and 0.1 p.p.m, s tandard solutions of IAA, GAs, KIN, and abscissic acid (ABA) respectively on the ordinate. The values 4- 12% are statistically significant.

Results

The effect of individual concentrations of the growth regulators on the development of the fruiting bodies on the synthetic medium can be observed in Table 1, and on the development of those growing on the medium contain- ing malt extract in Table 2. The results clearly show that the effect of the growth regulators differs not only with respect to the type of the regulator and the concentration used, but also with respect to the respective develop- mental stage under consideration. E. g. indole-3-acetic acid stimulates the dimension characteristics of the fruiting bodies i. e. the diameter of the

4 3 8 z. SL A D K Y , V. TICHY"

TABLE 1 S T I M U L A T I O N OF T H E FORMATION OF FRUITING- BODIES OF T H E FUNGUS Len t inus tigrimus ~T GIS, OWTH REGULATORS. ~ULTIV2KTION ON TH]~ SYNTH]~TIC MEDI'UM 51 D,~YS

Frui t ing body Number fresh weight Diameter Length

of fruit ing bodies product ion f rom of the cap of the stalk on cul t ivat ion one cul t ivat ion

cylinder cylinder [ram] [ram] [g]

Control 6.3 • 0.3 6.5 3= 0.3 22.6 3= 1.5 28.8 3= 1.4

IAA [ppm]

50 100 200 300

6.5 -4- 0.5 7.3 3= 0.3 28.9 3= 3.1 29.6 3= 2.0 5.0 3= 0.1 ++ 6.5 3= 2.8 31.3 3= 3.6 + 43.8 -4- 2.6 ++ 6.5 3= 0.5 6.8 3= 0.9 23.1 3= 3.7 27.6 3= 1.7 9.0 3= 0.1 ++ 8.1 3= 0.3 ++ 20.6 3= 3.1 27.8 3= 1.7

GAa 100 [ppm] 200

300 400

6.5 ~- 0.5 5.4 4- 1.4 18.6 4- 4.9 39.9 -4- 2.3 ++ 9.0 -4- 1.0 + 6.5 3=4- 1.8 24.6 3= 2.6 40.3 3=4- 4.0 ++ 9.0 4- 0.1 ++ 7.7 3= 0.8 20.8 3= 3.7 32.6 3= 1.9 5.0 3= 0.1 ++ 6.0 3= 1.2 22.5 3= 3.3 32.0 3= 3.0

K I N 100 [ppm] 200

300 4O0

7.5 • 1.5 7.2 • 4.2 27.3 3= 3.4 32.9 ~- 3.0 6.5 3= 0.5 5.1 3=4- 2.5 35.2 3= 1.8 ++ 37.5 3=4- 1.9 ++ 7.5 3=4- 0.5 5.1 3=4- 1.9 35.0 -4- 3.8 ++ 33.0 + 9.9 9.5 -4- 1.5 6.1 3= 0.4 13.6 4- 2.9 ++ 34.6 3=4- 4.7

Note: + stat is t ical ly significant, P < 0.05, ++ stat ist ically significant, P < 0.01

cap and the length of the stalk at rather lower concentrations near 100 p.p.m., whereas higher concentrations (300 to 400 p.p.m.) already retard their growth. As an exception, the diameter of the cap of the fruiting bodies growing on the medium with malt is not increased at any of the concentra- tions used but decreased by the lowest and by both the highest concentra- tions. In contrast to the mentioned characteristics, the number of fruiting bodies is the highest at higher IAA concentrations reaching the maximum at approximately 300 p.p.m. The weight of the fruiting bodies shows a similar dependence on IAA concentration as their number, however, most differences from the control are here smaller and statistically not significant. These results of the experiment with indole-3-acetic acid indicate tha t the in- hibition of the size of the fruiting bodies, i. e. essentially the elongation of individual hyphas, occurs simultaneously with the multiplication of fruiting bodies and with an increase in their fresh weight.

Gibbcrellic acid stimulates significantly on both media (as far as dimension characteristics are concerned) only the growth of the stalk at rather lower concentrations round 100 p.p.m. The diameter of the cap shows a not very pronounced vacillation around 100 % through the whole concentration range.

F O R M A T I O N OF F R U I T I N G B O D I E S OF LENTINUS 439

TABLE 2 STIMULATION OF THE FORMATION OF FRUITING BODIES OF THE FUNGUS Le~$~U8 Gl%OWTH REGULATORS. CULTIVATION ON 3~o SOLUTION OF MALT EXTRACT 51 DAYS

$igrinu8 BY

Frui t ing body Number fresh weight

of frui t ing bodies produc t ion Diameter Length on cul t ivat ion f rom one cul t ivat ion of the eap of the stalk

cylinder cylinder [ram] [ram]

[g]

Control 5.3 4- 0.3 3.8 4- 0.1 22.4 4- 1.5 28.7 + 1.7

I A A 25 8.5 4- 1.5 3.9 4- 0.5 10.2 4- 1.5 ++ 31.5 • 4.2 [ppm] 50 5.0 -4- 0.2 3.8 5=4- 0.2 19.0 4- 3.6 42.9 4- 2.6 ++

100 5.5 4- 0.5 4.4 4- 0.4 25.1 4- 3.4 36.1 4- 2.1 + 200 8.0 ~= 1.0 + 4.3 J= 2.0 27.5 J: 3.5 31.5 4- 1.3 300 13.0 =t= 1.0 ++ 6.1 4- 1.2 13.9 4- 2.2 ++ 29.0 4- 1.9 400 11.5 5= 2.5 + 2.8 5= 0.6 9.2 4- 1.2 ++ 22.3 5= 1.5 ++

GAs 25 7.5 • 0.5 ++ 7.1 4- 0.1++ 19.9 4- 2.4 39.8 4- 1.7 ++ [ppm] 50 6.5 4- 1.5 5.3 4- 1.8 23.9 4- 3.4 29.4 4- 2.4

100 6.0 4- 1.0 5.3 4- 0.2++ 19.0 :J: 9.5 46.6 4- 5.2 ++ 200 7.5 4- 2.5 4.9 4- 0.7 21.6 4- 2.4 30.4 4- 4.4 300 8.0 :t= 1.0 + 5.4 5= 0.7+ 20.4 d: 2.5 31.0 ~ 2.8 400 6.5 4- 1.5 4.8 5= 0.5 23.8 5= 3.4 53.3 4- 4.1

K I N 25 6.5 4- 1.5 3.3 4- 0.5 19.9 5= 2.6 25.0 4- 2.6 50 7.0 4- 0.1 ++ 4.6 4- 0.2++ 22.7 5= 2.2 29.3 5= 2.6

100 7.0 5= 1.0 4.8 5= 0.9 17.4 5= 2.5 33.7 5= 1.7 ++ 200 7.5 ~ 1.5 5.3 5= 0.3++ 22.3 5= 2.4 34.7 • 4.9 300 6.5 -~ 1.5 4.5 ~- 0.1++ 25.4 =- 2.1 37.4 + 2.0 ++ 400 7.5 + 0.5 ++ 3.5 ~ 0.1 14.2 r 2.5 ++ 30.5 ~ 3.0

Note: + statist ically significant, P < 0.05 ++ statist ically significant, P < 0.01

The number and the weight of the fruiting bodies growing on the medium containing malt increase through the whole range of concentrations (statis- tically significantly at 25 and 300 p.p.m, with a broader opt imum at the higher from both concentrations). On the synthetic medium both these characteristics show the maximum in the stimulation only at higher of both given concentrations (300 p.p.m.), even though no change in the weight of fruiting bodies is statistically significant. The dependence of the weight and of the number of fruiting bodies on GAa concentration is (equaLly as with IAA) on the same medium similar.

Kinetin increases the size of the fruiting bodies mainly at higher con- centrations (200 to 300 p.p.m.) with the exception of the diameter of the cap in the variant with malt extract where there is no expressive stimulation. The diameter of the cap even diminishes at the highest concentration. The number of the fruiting bodies is increased by a broad range of concentrations

440 z. SLADKS~, V. TICHY

with the increase being statistically significant only on the medium with malt. The course of the changes in their weight is in the main similar.

The tables show that opt imum concentration values occupy in most cases more or less broad zones. A more accurate determination of opt imum

Fig. 1.: Change~ in the level of the endogenous growth regulators in the stalks (S) and in the c a p s (C) of the fungus Lentinus tigrinus under the influence of optimum concentrations of t h e synthetic growth regulators (perpendicular rows of histograms: control, IAA 300, GAs 300, K I N 200). In the upper horizontal row of the histograms there are the results of the auxin bioassays, the middle row is formed by the values obtained with the bioassays specific for gib- berellin-like substances, and in the lower row t h e r e are the results of the cytokinin bioassays. Quantitative comparison of the stimulations and inhibitions is enabled by the respective standard solutions on the ordinate.

concentrations is made d i f f icu l tby a morphologie variability of the mycclium the skinlike formation of which makes difficult in some eases a uniform uptake of growth regulators from the medium. This is accentuated by the fact tha t at the end of the trial not all the harvested fruiting bodies reached the. same stage of development.

In the photograph (Fig. 2), an obvious difference can also be observed in the size of the fruiting bodies in individual concentration series of the st imulator used. I f for example the effect of IAA at higher concentrations (300 to 400 p.p.m.) causes an increase in the number of the fruiting bodies, then the development of their caps is later conspicuously retarded and the length of the stalk diminished. Gibberellic acid stimulates at lower con- centrations the length of the stalk bu t not the diameter of the cap. Kinetin promotes uniform growth and development of most of the fruiting bodies.

The results of the bioassays are presented arranged in histograms in Fig. 1. In the upper row results of the auxin bioassays are given, the middle row is formed by the values of the bioassays specific for gibbercllin-likc sub- stances, and in the lower row there are the results of cytokinin bioassays.

FORMATION OF FRUITING BODIES OF L E N T I N U S 441

Fig. 2. : Fruiting bodies of the fungus Lentinus tigr*nus growing on cellulose cylinders soaked with a 3% solution of malt extract (control above). The stimulatory influence of different con- eentrations of indole-3-acetic acid (IAA), gibberellie acid (GAa), and kinetin (KIN) on the shape and the number of fruiting bodies can be observed in the further parts of the figure.

442 z. SLADKY, V. TICH~2

The histograms in the first two perpendicular columns from left represent the level of the growth regulators in the stalk and in the cap of the fruiting bodies growing on the substrate with 300 p.p.m, of IAA. The exogenous application raises the level of auxins in the cap, decreases the level of gib- berellin-like substances in it and deepens the inhibitions in the stalk establish- ed by both the gibberellin and cytokinin bioassays. The changes caused by 300 p.p.m, of gibberellie acid are given in further two columns. They cause a slight increase in the level of auxins in the stalk and a total in- crease in the amount of gibberellins in the whole fruiting bodies. The Cy~o- kinin bioassay showed only minor changes. The last two columns of histo- grams represent the effect of exogenously supplied kinetin (200 p.p.m.). The exogenous supply conspicuously increased only the level of cytokinins, mainly in the stalk.

The results presented here show that growth regulators, when supplied exogenously to the cultivation medium of the fungus Lentinus tigrinus, can cause an increase in the level of the growth regulators of the same type in the fruiting bodies. Usually the ratio between the other growth regulators is also changed as well as the character of native inhibitions.

Diseussion

The transition from the vegetative growth of the mycelium to reproductio n is an important turning point in the life of fungi. The formation of the fruiting bodies of the fungus Lentinus tigrinus is controlled according to SC~WANT~S and I-IAGEMANN (1965) not only by a sufficient amount of the vegetative myeelium but also by a decreased CO2 concentration in the medium and by adequate illumination. Although our method of cultivation corresponded sufficiently to these conditions, we could nevertheless find in our earlier experiments some underdeveloped pointed fruiting bodies lacking the cap. The bioassays have shown that some substances with inhibitory character with changing activity were present in such primordia (RYP~6Ex and SLADK~7 1972).

While the data on the effect of growth regulators during the vegetative growth of the myeelium are numerous (GRUEN 1958, PHI~EY and WEST 1960, GOOALA 1970), the knowledge about their influence on the reproductive development is more scanty (SZABO et al. 1970). Little is also known about the occurrence and the character of inhibitory substances which accumulate in some stages of development of the fruiting bodies (PARK 1963).

The analyses show that the exogenous application of the growth regulators leads to an increase in the level of the endogenous growth regulators of the same typ with a simultaneous change in the ratio between the other growth regulators, and that native inhibitions are overcome in this process. The positive effect of all the three types of the growth regulators indicates that the inhibitory barriers are labile in the mycelium.

The morphological distinction of the fruiting bodies developing under the influence of the individual types of the growth regulators indicates that these substances are specifically involved in the differentiation process of the fruiting bodies. Thus, exogenously supplied IAA increases expressively the number and the weight of the fruiting bodies, but decreases the diameter

FORMATION OF FRUITING BODIES OF L E N T I N U S 443

of the cap and the length of the stalk. The growth of both of these parts of the fruiting bodies is, on the contrary, increased by lower IAA concentrations. That means that there exists an indirect relationship between the number of formed fruiting bodies and their growth. This relationship is not so ex- pressive in the variants with GA3 and KIN and is partly conditioned by the nature of the medium.

The results presented in this paper completed by a conception about the dynamics in the pattern of the growth regulators during different develop- mental stages of the fnngus underline their significant position in the system of the regulation.

Re[erenees

BENTLEY, J. A., HOVSLEY, S.: Bioassay of plant growth hormones. - - Physiol. Plant. 7 : 405 -- 419, 1954.

FX~rLAND, B., WAR~I~G, P. F.: Effect of gibberellic acid on hypocotyl growth of lettuce seedlings. - - Nature 185 : 255~256, 1961.

GOGALA, N. : Einfluss der natiirlichen Cytokinine yon Pinus sylve,tris L. und anderer Wuehsstoffo auf das Myzelwachstum yon Boletus edulis var. pinicolua VI~r. - - r bet. Z. l l 8 : 321--333, 1970.

G ~ N , H. E.: Auxin and fungi. - - Armu. Roy. Plant Physiol. 10 : 405--440, 1958. OSBORNE, D. J . , McCALLA, D. R.: Rapid bioassay for kinetin and kinetins using seneseing leaf

tissue. - - Plant. Physiol. 36 : 219--221, 1961. PA~K, D.: Evidence for a common fungal growth regulator. - - Trans. brit. myeol. See. 46 :

541--548, 1963. PHrgNEY, B. O., WEST, Cm A.: Gibberellins as native plant growth regulators. - - Annu. Rev.

Plant Physiol. 11 : 411--436, 1960. RX~-6EE, V., SL~-DX'2, Z.: The character of endogenous growth regulators in the course of

development in the fungus Lentinus tigrinus. - - Mycopathol. Myeol. Appl. 46 : 65--72, 1972. RYP-~6EK, V., SL_~DK'~, Z.: Relation between the level of endogenous growth regulators and the

differentiation of the fungus Lentinus tigrinus studied in a synthetic medium. - - Biol. Plant. 15 : 20--26, 1973.

SCHWA~TES, H. O., HAOE~A~-~, F.: Untersuchungen zur Fruehtk6rperbildung bei Lentinus tigrinus BULL. - - Ber. deutsch, bet. Ges. 78 : 89--101, 1965.

S z ~ 6 , L., POZS~R, B. I., KOTA, M.: Cytokinin activity of the fruiting body of Coprinus micaceu$ F~. - - Aota Agr. Aead. Sci. hung. 19 : 402--403, 1970.

URAYA~A, T.: Stimulative effect of certain specific bacteria upon mycelial growth and fruit body formation of Agaricus bisporus (LANOE) Srso. - - Bet. Mag. (Tokyo) 74 : 56--59, 1961.

Z. SLADK~ r, V. TICHq, Katedra biologie rostlin pHrodov~dcck~ fakulty University J. E. Purkyn~, Brno: St imulaee tvorby plodnle houby Lentinus tigrinus (Bull.) Fr. rfistov~ml re9u-

16tory. - - Biol. Plant. 16 : 433--443.. 1974.

Rflstov6 stimul~ttory kyselina ~-indolyloetovgL (IAA), kyselina giberelovs (GAa) a kinetin (KIN) v rflzn:~ch koncentraclch byly pou~ity ke stimulaci vzniku a dalw rflstu plodnic houby Lentinus tigrinus. Vegetativnl mycelium houby bylo kultivov&no na celulosov:~oh vs nashkl~eh syntetick~m ~ivn~rm roztokem nebo 3 % roztokem sladov~ho v~ta~ku. Kdy~ mycelium porostlo povreh vgdeSkfl, probfhala dal~/kultivaee v odstupflovan:~eh koncentracfeh uveden~ch stimul~torfl. Podle poStu plodnic jsme zjistili, ~e optims koncentrace IAA a GA3 v obou kultivaSnfeh mediich je 300 ppm. U kinetinu byla optims koncentrace 400 ppm.

Jednotliv6 typy stimulgttorfl ovlivnily eharakteristieky i tvar plodnie a m~nily v nich p~iro- zenou hladinu endogennfch regul&torfl. PHdgmi IAA do media zv~w hladinu auxinov~ch lhtek v klob0uku. P~itomnost giberelov~ kyseliny nebo kinetinu v mediu se projevila zv:~enlm hladiny obou t~chto l&tek v plodnielch.