ROCZNIKI GLEBOZNAW CZE, T. X X X II, NR 3, W ARSZA W A 1981
R OM A N PA C H L E W SK I, ELŻBIET A C H R U SC IA K
EFFECT OF MINERAL SOURCES OF PHOSPHORUS ON THE GROWTH OF ECTOMYCORRHIZAL FUNGI
D ep a rtm en t of S o il S c ie n c e and F ertiliza tio n , F o restry R esearch In stitu te, W a rsza w a -S ęk o cin I n stitu te of S o il S c ien ce W arsaw A g ricu ltu ra l U n iv e r sity
IN T R O D U C TIO N
One of the im portant physiological effects of m ycorrhiza in the ecosystems is the phosphorus turnover intensification (1-3, 5-10, 13). It has been proved th a t the plants growing in the symbiosis w ith m ycor- rhizal fungi absorb m uch m ore of this elem ent than control plants. Differences occur p articu lar distinctly w hen in the su bstrate hardly- soluble phosphorus compounds are contained. This phenomenon can be explained by forming as metabolism products organic acids, solving m any m inerals and thus m aking available to plants elements contained in them.
The initiation and intensification of the m ycorrhizal infection are favourably affected by the N and P deficiency. However, an acute phosphorus deficiency in soil is accompanied by an inhibition of forming m ycorrhizae, because, among other things, of the fact of participation of this elem ent in arrangem ent of active enzymes in carbohydrate tra n s formations. A low level of sugars in the plant results in a lack of forming m ycorrhizae [7].
In view of the m ineral fertilization of nurseries and forestands, ap- v plied wider and w ider in the forestry, it is necessary to recognize the reaction of ectom ycorrhizal fungi to the above m easure, particularly in case of phosphorus fertilization. This question is of a particu lar im portance in view of a quick fixation of the fertilizer phosphorus in soil, w hat reduces to a considerable extent its utilization by plants. In acid soils, to w hich m ost forest soils belong, phosphorus is im mobili zed assuming the forms of iron and alum inium phosphates. In this con nection the estimation, to w hat degree symbiotic fungi are able to utilize these P sources and thus m ake this elem ent available to plants, is of significant importance.
76 R. Pachlewski, E. Chruściak
The subject of investigations is the attem p t to determ ine under laboratory conditions the utilization by chosen fungi species phosphorus
fertilizer tested and hardly-soluble AIPO4 and F e P 0 4. This stage of
observations was regarded as a prelim inary contribution to works to be carried out on the m ineral NPKCa fertilization effect on the sym biotic activity of fungi in the m ycorrhiza test under control experim ent conditions.
The results of analogic investigations concerning nitrogen fertilization are presented in the work carried out in 1978 (12).
M A TER IA L A N D M ETH ODS
C u l t u r e s o f f u n g i . The respective observations concerned fungi species (Table 1) originating from the collection of the Laboratory of Biology of Forest Soils, F orestry Research Institute at Sękocin.
T а Ъ 1 0 1 F u n g i s p e c ie c exam ined S p e c ie s C u l t u r e c o l l e c t i o n N o . ;'c t o m y c o r r h i z a l t e îï t w i t h p ino /11/ A m anita v e rn a / B u i . e x F r / P o r s . ex V i t t . 0141 +
Cenococcum g ra n ifc rr a e /S o w ./ F e r d . e t Winge 3543 +
4947 r o t t e s t e d
R hizopogon l u t e o l u a F r . C211 +
S u i l l u e b o v in u s / L . ex F r , / 0 . K untze 1941 +
S . l u t e u s /L . ex F r . / S ,F , Gray 0112 +
T ric h o lo m a a lb o b ru n n e u a / Р е г з . ex F r . / Kwamer 1951 +
P h o s p h o r u s s o u r c e s . In the experim ents pulverized and
granulated superphosphate as well as Ca(H2P0 4). H20 in pure form,
alum inium and iron phosphates were used. F e P 04 is alm ost insoluble
w ithin the pH lim its of 2-6, and AIPO4— of 4.5-7.0. The solubility of
both compounds increases along w ith decreasing acidity. As control
KH2PO4 was applied.
C u l t u r e s u b s t r a t e s . Phosphorus compounds in the suitable concentrations were added w ithin an exchange to the basic substrate of the following composition : glucose — 20 g, maltose — 5 g, ammonium ta rtra te — 0.5 g, M gS04. 7 H20 — 0.5 g, iron citrate (iVo solution) —
0.5 ml, Z n S 04 (0.2°/o solution) — 0.5 ml, thiam ine — 50 jbig, agar — 15 g,
distilled w ater — 1 0 0 0 ml.
N utrien t media poured into the P e tri dishes were centrally inocul ated by mycelium discs of 0.9 m m in dia, cut by sterile cork-borer out cf a suitable 14-21-day culture on the substrate as above, in w hich
E ffect of phosphorus on fungi 77 T a b l e 2 B a t e s o f p a r t i c u l a r F compounds Variant No. Compound F e r t i l i z e r r a t e i n kg P 20^ p e r h e c t a r e Compound q u a n t i t y i n th e n u t r i e n t medium mg/1 P q u a n t i t y in th e n u t r i e n t medium m g/1 1 kh2po4 - 1000 220 2 p u lv e r iz e d s u p e r p h o sp h a te 30 58 4 3 p u lv e r iz e d su p e r p h o sp h a te 60 116 8 4 p u lv e r iz e d s u p o rp h o sp h a te 120 232 1С 5 p u l v e r i z e d su p e r p h o sp h a te - 3190 P. 20 6 g r a n u l a t e d s u p e rp h o sp h a te 30 52 4 7 g r a n u l a t e d su p e r p h o sp h a te 60 104 Û 8 g r a n u l a t e d s u p e rp h o sp h a te 1*0 208 16 9 g r a n u l a t e d su p e r p h o sp h a te - 2860 220 10 Ca/H2P 04/ 2 ,H20 30 13 4 11 Са/Н2Г04/ 2 .К 20 60 36 8 12 Ca/H2P04/ 2 . 4 20 120 72 16 13 Ca/H2P04/ 2 „H20 - 931 220 14 a ip o4 n o t a p p l i e d 32 8 15 a ip o4 n o t a p p l i e d 64 16 16 a ip o4 n o t a p p l i e d 880 220 17 PeP04 n o t a p p l i e d 40 8 18 PeP04 n o t a p p l i e d 80 16 19 FePO,4 n o t a p p l i e d 1100 220 20 0 0 0 0
the P source was KH2P 04 (Pp). The incubation ra n at 25°C for 14 days.
Each experim ent v aria n t was carried out in two replications. An. ad ditional com bination constituted the subtrate com pletely lacking phos phorus and the control v aria n t on the Pp n u trien t medium. The grow th of cultures was determ ined on the basis of the mycelium diam eter m easurem ent.
R ESU L TS
The effect of particular phosphorus sources on the grow th of ecto- m ycorrhizal fungi appeared to be easier for estim ation for relatively quick growing species, such as Am anita verna, Rhizopogon luteolus, Suillus bovinus, S. luteus, Tricholoma albobrunneum. On the other hand, in both slower-growing Cenococcum graniform e strains these differences are less distinct. On th e basis of observations it was successful in proving th a t two species — A. verna and R h . luteolus, show g reater demands of phosphorus as compared w ith rem aining fungi, which are ra th e r toleran t to the lack of this elem ent (Figs 1-7).
78 H. P a ch lew sk i, E. C hruściak
F ig. 1. M y celiu m d ia m eter am ou n t of th e com pound in m g per l litre of su b strate : I — 1000, 2 — 58, 3 — 116, 4 — 232, 5 — 3190, 6 — 52, 7 — 104, 8 — 208, 9 — 2860, 10 — 18, 11 — 36, 12 —
72, 13 — 931, 14 — 32, 15 — 64, 16 — 880, 17 — 40, 18 — 80, 19 — 1100, 20 — n o p h o s p h o r u s
tested in view of ra th e r great differences in results, not enabling any explicit general estimation.
Amanita verna. O ptim um for this fungus proved to be the substrate containing phosphorus in the form of KH2 P 0 4. The w eakest grow th was found on substrates w ith all rem aining phosphorus compounds in the concentration corresponding w ith 1000 mg/1 KH2P 0 4 and on the substrate completely lacking P. The equal effects w ith respect to grow th were obtained on substrates containing the lowest pulverized and granulated superphosphate amounts, 8 mg/1 F e P 0 4 and 16 mg/1 A1P04. Between p articu lar rates of different compounds (except for the highest) not especially high differences were observed. The application of a pure form of Ca(H2P 0 4)2. H20 did not resu lt in any b etter grow th effects th an at technical preparations.
Cenococcum graniforme. Both strains (Polish 3543 and A merican 4947) were characterized by approxim ate reaction. A sim ilar grow th both on the Pp n u trien t m edium and on th a t w ith a complete P lack is to be stressed. The best grow th showed the 3543 strain on substrates
È ffëct of phosphorus on fu n g i 79
F ig. 4. C om parison of th e g ro w th rea ctio n of ectom ycorrh izal fu n g i to p h osphorus com p ou n d s — p u lv erized su p erp h osph ate, A 1 P 0 4 and F e P 0 4 (m edium rates)
80 R. P a ch lew sk i, E. C hruściak
Fig. 6. T r ic h o l o m a a lb o b r u n n e u m grow th on th e n u trien t m ed iu m w ith an ad d ition of p u lv erized su p erp h osph ate in th e am ou n t of 116 mg/1
Fig. 5. T r ic h o l o m a a lb o b r u n n e u m g row th on the n u trien t m ed iu m w ith an ad d ition of p u lverized su p erp h osph ate in the am ount of 3190 mg/1
E ffect of phosphorus on fungi 81
Fig. 7. R hizopogcm lu te o lu s grow th on the n u trien t m ed iu m w ith an ad d ition of p u lv erized su p erp h osph ate in th e am ount of 3190 mg/1 — le ft p la te, 116 mg/1 —
righ t p la te
containing Ca(H2P 04) 2 in both pure and technical form. The 4947 strain
utilized omewhat better AIPO4 and F e P 0 4. A worse grow th was obser
ved, like in A. verna, on substrates w ith the P concentration correspon
ding w ith KH4PO2.
Rhizopogon luteolus. It is the species reacting negatively to a complete lack of phosphorus and the highest concentrations of the compounds tested. The best P source proved to be m edium rate of g ranulated superphosphate and, w hat is of interest, of alum inium and iron phos phate. The fluctuations in the grow th on substrates w ith rem aining rates of particular compounds can be regarded as lying w ithin the error limit.
An attention deserves the phenomenon of differentiation of the pig m entation of cultures depending on the P source. In the culture sub
strate ; at application of Ca(H2P 04) 2 the mycelium was of flesh colour,
at th a t of A I P O 4 of ru sty colour, at th a t of F e P 04 — of dark-brow n
colour.
Suillus bovinus. This fungus is to leran t to the phosphorus level, w hat m anifests itself in an almost identical grow th on the Pp n u trien t medium
containing KH2P 04 as on the phosphorus-lacking n u trien t medium. It
reacted, best except for potassium phosphate, to chemically pure
Ca(H2P 04) 2 H20 applied in a medium rate. Four rem aining compounds
in lower concentrations were utilized identically. Surprising is the fact
82 R. Pachlewski, E. Chruściak
that all compounds applied, except for Са(Н2Р 04) 2 Н20 , particularly
when applied in a medium rate, showed, on the whole, the reaction some w hat inhibiting the grow th as compared w ith the phosphorus-lacking
substrate and containing KH2P 0 4.
Suillus luteus. Ca(H2P 04) 2 H20 in chemically pure form proved for
this fungus to be a compound affecting at best the grow th (at the lowest
rate), sim ilarly as KH2P 0 4. It is relatively well to leran t to a lack of
phosphorus ; on the su bstrate w ithout phosphorus it grew only slightly worse than on Pp, not showing any negative reaction when grown on rem aining substrates. The higesht rates of compounds used in the expe rim ents led to a grow th inhibition of the mycelium.
Tricholoma albobrunneum. The best among P sources proved to be for this fungus pulverized superphosphate, p articu larly w hen applied in a m edium rate. Remaining compounds in any rate (except for the highest, which have inhibiting effect) were at least equally favourable
for the fungus growth, as KH2P 0 4.
D IS C U S S IO N
In connection w ith a lim ited num ber of works analogic to ours, it was difficult to find a reference point for the results obtained in the present work. More frequent were, instead, the experim ents concerning definite symbiotic arrangem ents of m ycorrhizae.
In the experim ents of M ejstrik and K r a u s e [5] on phosphorus assimilation by m ycorrhizal associations of Pinus radiata and Suillus luteus or Cenococcum granijorme differences were found as compared w ith control plants in the assimilation intensity of P (in the form of
H3P 04 and organic P in humus) depending of fungal symbionts. Thus
the seedings ionoculated w ith S. luteus assimilated by 45% more P than control plants, while those inoculated w ith C. granijorme — only by 13% more P. These differences can be explained by a different anatomic stru c ture of the mycorrhiza — the phosphorus-assim ilating apparatus — b u t m ainly by the differentiation of metabolic processes depending on phy siological functions of these fungi. Moreover, the authors have found that
C. granijorme assimilates P32 more efficiently from organic compounds.
These results correspond inasm uch w ith ours th a t they explain to some extent an almost complete lack of differences in assimilation of the m ineral compounds applied by C. granijorme. It m ay be supposed th a t introduction of the v arian t w ith an organic P source would give a con trastive picture.
On the fact th a t for m ost fungi tested the Ca(H2P 04) 2 H2 0 concen
trations applied, (lower th an those of KH2P 0 4) were sufficient, a certain
Effect of phosphorus on fungi 83 comparing the grow th of m ycorrhizal and control plant in soils
containing increasing Ca(H2P 04) 2 H20 am ounts has been found th a t
m ycorrhiza shows a m ortality tendency at the concentration of this
compound of 1.0 g per kg of soil. A t the ra te of 6 . 0 g per kg of soil
m ycorrhiza perished and also only a p a rt of the fungal inoculum su r
vived in soil. In our experim ents the ra te of Ca(H2P 04) 2 of 931 mg per
1 1 of n u trien t medium resulted also in a lim ited grow th of the fungi
tested, but b etter than analogic concentration in both superphosphate forms. This phenomenon was observed in all fungi tested.
C O N C L U SIO N S
1. The present experim ents have proved th a t there are differences in phosphorus demands of m ycorrhiza fungi species tested. The highest dem and showed A . verna and Rh. luteolus, the rem aining species charac terizing by a fairly well tolerance to the deficiency of this elements.
2. K H2P 04 applied as control at the rate of 1000 mg per 1 1 of the
n u trie n t m edium proved to be an optim um P source w ith reg ard to th e compound kind and qu ality of P delivered to A. verna, utilized sim ilarly well as other compounds by Rh. luteolus as well as by S. bovinus and S. luteus sim ilarly well as Ca(H2P 04) 2 H 20 it is som ew hat worse utilized by both C. graniform e strains than rem aining compounds (except for the highest rates).
3. Pulverized and g ranulated superphosphate applied in the am ount corresponding w ith medium or low fertilization rates, resulted under conditions of the present experim ent in a positive growing reaction of ectom ycorrhizal fungi tested.
4. The tolerance of low P concentrations on substrates, found in 5 strains, proved th a t it can constitute one of the reasons th a t the com
pounds applied a t the rates m uch lower th an th a t of KH2P 0 4, were
equally good or even b etter phosphorus source.
5. The chem ically pure Ca(H2P 04) 2 H 20 showed, as compared w ith
pulverized and granulated superphosphate, a b etter grow th reaction only in both Suillus species : S. bovinus and 5. luteus, although when applied at the highest rates, it caused a weaker grow th inhibition of all fungi tested.
6. The experim ent resu lts proved explicitly the ability of ectomy
corrhizal fungi for A1P04 and F e P 04 utilization as a P source. This
phenomenon, p articu larly distinct in in A. verna and Rh. luteolus species, leads to some reflections as to th e assim ilation m echanism of these compounds, while taking into consideration th a t at the reaction applied in the experim ents (pH 5.5) these compounds rem ained unsolved. It can be supposed th a t these compounds can be assim ilated by fungi in the form of chelates [4].
84 R. Pachlewski, E. Chruściak
7. Isolates belonging to the same species (two C. graniformis strains) or the same genus (S. bovinus, S. luteus) proved a certain convergence in reaction to the phosphorus sources applied.
8. Pulverized superphosphate applied a t the rate corresponding w ith
KH2P 04 caused a total grow th inhibition of all strains tested (Fig. 3) ;
on analogic substrate containing granulated superphosphate only the grow th of Rh. luteolus and S. bovinus (besides very weak) was observed, whereas all strains grew well on the n u trien t medium containing even
the highest Ca(H2P 0 4)2, H20 , A1P04 and F e P 04 rates (Fig. 3) the
concentrations of the above compounds equal to th a t applied in the practical forestry, giving b etter grow th effects.
9. On the basis of litera tu re data cited and the experim ents carried out w ithin the fram ew ork of the present study, one can conclude th a t a certain sim ilarity would exist as regards requirem ents of am ount, kind and form of P in symbiotic fungi in vitro and in situ.
REFEREN CES
[1] D o m i n i k T.: S tu d y on m icotrop h ism of com m on spruce. P race IB L 209, 1961, 39-102.
[2] H a c k s k a y l o E. : M ycorrhizae. W ash in gton 1971. [3] H a r l e y J. : B iology of m ycorrhiza. L ondon 1959.
[4] H u t n e r S. H. : In organ ic n u tritio n . A n n . R ev. M icrob. 26, 1972, 313-346. [5] M e j s t r i k V. K., K r a u s e H. : U p ta k e of 32P by P in u s ra d ia ta roots
in o cu la ted w ith S u illu s lu te u s and C en o co ccu m g ran iforrn e from d iffe r e n t sou rces of a v a ila b le ph osp h ate. N e w P h y to l. 72, 1973, 137-140.
[6] M e j s t r i k V. K. : The effect of m ycorrhizal in fection of P in u s silv e s tr is and P icea a b ie s b y tw o B o le tu s sp ecies on th e a ccu m u la tio n of phosphorus. N e w P h y to l. 74, 1975, 455-469.
[7] M e l i n E. : P h y sio lo g y of m ycorrh izal rela tio n s in p lan ts. A nn. R ev. P la n t P h y sio l. 4, 1953, 325-345.
[8] M о s s e H. : P lan t grow th resp onses to vesicu lar-a rb u scu la r m ycorrhiza. N ew P h y to l. 72, 1973, 127-136.
[9] M о s s e B., H a y m a n D. S., A r n o l d D. J. : P la n t gro w th resp on se to v e sic u la r -a r b u sc u la r m ycorrhiza. V. P h osp h oru s u p tak e by tree p la n t sp ecies from P -d e fic ie n t so ils la b elled w ith 32P. N ew . P hytol. 72, 1973, 809-815. [10] M u r d o c h C. L., J a c k o b s J. A. , G e r d e m a n J. W. : U tiliz a tio n of
phosp h oru s sou rces of d iffe r e n t a v a ila b ility by m y corrh izal and n o n -m y co r- rh izal m aize. P la n t and S o il 37, 1957, 329-334.
[11] P a c h l e w s k i B., P a c h l e w s k a J. : S tu d ies on sy m b io tic p rop erties of m ycorrh izal fu n g i of p in e w ith th e aid of th e m ethod of m ycorrh izal sy n th e sis in pure cu ltu res on agar. W arsaw 1974.
[12] P a c h l e w s k i R., C h r u ś c i a k E., Z a w i s t o w s k a T. : N itro g en u tiliz a t ion by m ycorrh iza fu n g i from v a rio u s form s of fe r tiliz e r s applied in the fo rest fe r tiliz a tio n . Rocz. gleb ozn . 29, 1978, 101-110.
Effect of phosphorus on fungi 85
R. PACHLEW SKI, E. CHRUSCIAK
W PŁY W M IN E R A LN YC H ŹRÓDEŁ FO SFO R U N A W ZRO ST GRZYBÓ W EKTOM IKORYZO W YCH
Z akład G leb o zn a w stw a i N a w o żen ia
In sty tu tu B ad aw czego L eśn ictw a , W a rsza w a -S ęk o cin 'In stytu t G leb o zn a w stw a A R w W arszaw ie
S t r e s z c z e n i e
P o ró w n y w a n o w zro st sied m iu szczep ów grzyb ów ek to m ik o ry zo w y ch (rep rezen tu ją cy ch 6 gatunków ) na podłożach zaw ierających różne zw iązki m ineralne fo sforu: su p erfo sfa t py listy , gra n u lo w a n y , C a(H2P 0 4)2H 20 w form ie chem icznie czy stej, A 1 P 0 4, F e P 04 i kontrolnie KH2P 0 4. N aw ozy zostały użyte w daw kach: m in im a ln ej, śred n iej i m a k sy m a ln ej, o d p o w ia d a ją cy ch 30, 60 i 120 k g /h a P2Os oraz k o n cen tra cji P o d p ow iad ającej ilo ści tego p ie r w ia stk a w 1000 m g K H2P 0 4. K ryteriu m o cen y p rzyd atn ości danego zw iązk u sta n o w ił p om iar śred n icy g rzyb n i w y r o słe j na pod łożach z o d p o w ied n im i źród łam i P.
Na p o d sta w ie p rzep row ad zon ych d o św ia d czeń u stalon o n a jw ię k sz e za p otrzeb o w a n ie na fo sfo r u A m a n ita v e r n a i R h izo p o g o n lu te o lu s. P o zo sta łe g a tu n k i m ożna o k reślić jako to leru ją ce n isk ie stężen ie fosforu , a n a w e t jego brak. K H2P 0 4 oka zał się op ty m a ln y m źródłem P co do rodzaju i stężen ia tego zw ią zk u ty lk o dla A . v e rn a . C a(H2P 0 4)2 w postaci czystej oraz techn icznej b ył p rzysw ajan y przez b ad an e grzyb y w d aw k ach n a w o zo w y ch sto so w a n y ch w p ra k ty ce leśn ej, przy czym p rzyd atn ość każdej z form dla różn ych g rzyb ów k szta łto w a ła się od m ien n ie.
D o św ia d czen ia w y k a za ły , że b adane grzyb y e k to m ik o ry zo w e zd oln e są do w y k o rzy sty w a n ia z w ią zk ó w fo sfo ru z trudno rozp u szczaln ych — A 1 P 04 i F e P 0 4, przy czym dla g a tu n k ó w A . v e r n a i Rh. lu te o lu s sta n o w iły one bardzo dobre źródło fosforu .
W szy stk ie za sto so w a n e zw ią zk i, a g łó w n ie su p erfo sfa t p y listy , u ży te w stężen iu P od p o w ia d a ją cy m 1000 m g /l K H2P 0 4, pow odow ały znaczne ograniczenie w zrostu te sto w a n y c h izolatów .
Prof. d r R o m a n P a c h le w s k i I n s t y t u t B a d a w c z y L e ś n i c t w a w Sęk o c ln ie , 05-550 R a s z y n
