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Phosphate-solubilizing microorganisms of a lateritic soil
Zbl. Dakt. II. Abt. 136 (1981),487-492
[University College o f Agriculture, Microbiological Laboratory, Calcutta University, Calcutta, India]
Phosphate-Solubilizing Microorganisms of a Lateritic Soil D. Effect of Some Tricalcium Phosphate-Solubilizing Microorganisms on Available Phosphorus Content of the Soil") S.
BANIK
and B. K.
DEY
Summary Five effic ien t phosphate-solubilizing microo rganisms isolated from a lateritic soil (Typic Ochragualf), L OBI' Bacillue [irmu« (B-7647), LOB" a mixed culture of Bacillus circulan8 and B. 8ubtili8 (B-7648), LCS., a Streptomgce» sp., LCF3 , an unreported species of A8pergillu8, and LOF., A. niger van Ti eghem, were inoculated in the said soil with rock phosphate (RP), farm yard manure (FYM), FYM + HP, rice straw (RS), and RS + RP in a flask culture experiment to study their role in impro ving the available phosphorus status of the soil under naturals non-sterile, and partially sberitized con d it ion s. The aspergilli L OF 3 and LOF. and the Bacillus LCB I showed their efficiency in solubilizing rock phosphate in broth cult ure. The aspergilli were also efficient cell ulose decomposers. Partial sterilization by autoclaving of the soil had no significant effec t on available phosphorus con ten t of the soil, irrespecti ve of a n y ino culation. Inoculation of L OF. and LCS. increased the available phosphorus con ten t of the unmanured soil. Application of manures, es pecia lly that of ri ce straw, lowe red the con ten t, wh er eas inoculation of LOF3 and LCS. with FYM a nd that of LOB, with FYM + HP in cr e ased the a vailable phosphorus con tent of t he soil. Howe ver, a s ignif ica n t effe ct of inoculation was obta ined only with L OF 3 an d L CFt in the respective cases m entioned.
Zusammenfassung Eine m L ate r-it (t yp. Ochragualf) wurde Rohphosphat, Stalldung und Reisst r oh e in zeln und in K ombination zugesetzt. Von flinf a us di ese m Boden isolierten Mikroorganismen erh oht en Aspergillu 8 n ige r und S treptomgces s pec. den Ge halt an verfiigbarem Phosphat des unbehandelten Bod en s. AHe Zu satze a u 13er Rohphospha t verringerten das ver fiigba re Phosphat, insbesondere R eisst ro h . Signifikante Erhohungen e rgab di e Beimpfung mit A. nig er und A spe rqillu» spe c. Irn einzelnen wirkte Beimpfung mit d en beiden Aspergillen in del' Variante Stalldung - ni cht steril und Beimpfung mi t eine r Mischkultur von B acillue ci rcul an8 und B. 8ubti li 8 in del' Variante Stalldung + R eisstroh - ni cht steril deutlich er h ohe n d au f den Gehalt an verftlgbarem Phosphat.
Among the different groups of phosphate-solubilizing microorganisms present in soil (GOLEBIOWSKA 1956), the performance of the group dissolving tricalcium phosphate in excess of the requirement appears to be ecologically and agronomically very important under Indian conditions, as most of the soil and natural phosphorus are lock ed in tricalcium phosphate. Carbonaceous materials play are crucial role when biochemical activity of these chemo-organotrophs is concerned . Supplementation of natural rock phosphate with organic manures enhances the proliferation of the phosphate solub ilizers and, as a consequence, intensifies solubilization of insoluble inor ganic phosphates in the soil (SI NKHA 1970; FARDEAU and G UIRAURD 1971). Bacteri1) P art of t he Ph.D. T hesis, su b m it ted b y the fir st author t o the U n ive rs ity of Ca lc ut t a in 1979.
488
S. B ANIK a nd B. K. D EY
zation with t he resp onsible microflora accent ua tes t he process (HAYMAN 1975). The present work was undertak en in order to obtain information regarding t he accumulation of a vailable phosphate in a lat eri ti c soil as influenced by t he inoculation of some efficient phosphat e-solubilizing microorganisms, isolated fr om the soil, in absence and presence of organic manures with or without rock phosphate under partially sterilized a nd non- sterilized na tural cond it ions.
Materials and Methods Source of isolates T he cult ures used in t his st ud y were selecte d from those isolat ed from a lateritic soil (Typic Ochragu alf, R UDRA 197 5) in P IKOVSKA IA'S (1948) suc rose ca lc ium phos phate aga r pl ates, showing hi gh phosphat e-solubilizing ab ility (BANIK and DEY 1981). T hese were L CBI' B acillus [irmu« (B -764 7); L CB~, a mixed cult u re of Bacillus circulans an d B. subtilis (B· 764 8); L CS 4, Strep tomyces sp. ; L CF s' an unreported sp ecies of Asperqillu»; an d LCF4 • Aspergill us niger van Tieghem (BANIK a n d DE Y 1981). The cultures we re maintaine d on the said PIK OVSKAIA' S (194 8) medium. The cu lt ures were tested fo r t hei r a b il ity of solubilizing rock phosphate an d de composing cellulo se, as ro ck phosphate and organic mat ter, cont ain ing appreciable amount of cell ul osic materials, we re included in the treatments wit h so il in t he su bseq uent experiment, and H 2S-producing ability is one of t he mean s of liberating phosphate from F eP0 4 present in t he soil.
Solubilizati on of rock phosphate Phosp h a t e-di ssol ving abili t y of t he isolates fr o m rock phosphat e was deter mine d in m od ified PIKOVSKAIA'S (1948) broth , containi ng 20 g ro ck phosphate, h a ving a n eq uivalent a mount of phosphor us as in 5.0 g CaS (P 0 4)2' T he p rocedures were t he sa me as foll owed b y B ANIK a n d DEY (198 1).
Decomposition of cellulose Ability to decompose cellulo se, i.e., to utilize cell ul ose as sole so urce of ca rbo n an d energy, was t est ed in Du bos' me d ium (ALLEN 195 7), us ing filter pape r strips in triplicate. The method of ino culat ion of t he isol ates was sim ilar to t hat follo wed by BANIK and DEY (198 1), T he tubes were incub ated at 30 ± 1 °C for one m onth. T he extent of deterioration of t he fil t er pa per str ips indicat ed the cell ulo se-decom posing abifit.y.
Production of H 2S T he ability for p roducing H 2S was t ested b y bl ackening of lea t acetat e-filter p a per str ips as descr ibe d by SALLE (196 7).
Avail abl e pho sphorus content of t he soil as affected b y inoculation of the isolates F or est im at ing the abilit y of t he isolates for bringing una vailable phosphorus to an avail able state in soil, 10.0 g lots of the sa me soil (BANIK a nd DEY 1981) h arbouring the isolates, were taken in 100 ml conical flasks with the foll owing treatments: (a) Untreated cont ro l ; (b) Rock phosphate (RP) , 30 kg p . ha - I ; (c) Farm y ar d manu re (FYM), 40 kg N· h a- I ; (d ) F YM, 40 kg N + RP, 30 kg p . h a - I ; (e) Rice straw (RS) , 40 kg N· h a - I ; and (f) RS, 40 k g N + RP, 30 kg p. ha: ", The mo isture le vel was kept at 100 % wa t er h olding cap ac it y, since the su bs eque nt experiment was proposed to be done on ri ce rhi zosphere under flooded con dit ion . F or eac h t reat me n t 36 fl asks we re ke pt. Out of t hese , 18 flasks we re autoclaved at 15 1b stea m pressure (121 ec ) for 30 m inutes on two con sec ut ive days, i.e. , so-called pa rtial steril ization , with a view t o co m pa re t he activity of the ino culated isola tes unde r a minimal microbia l co m pet it ion in soil. Partially steril ized a nd n on-st e ril e flask s we re inoculat ed with t he isolates in triplicate peI' manurial treatment. The proce dure of in ocul at ion was t he same as t hat ad o pte d b y BANIK an d DEY (198 1). The fl ask s were incu bated at 30 ± 1 °C. T he moisture level was maintaine d at 100 % wat e r h old in g ca pacity b y aseptica lly a dd ing sterile d istilled water e ve r y alternate d a y. Aft er 15 d a ys of incubat ion, t he available p ho sp horus of t h e fla sk content was est im at ed , following BRAY a n d K URTZ' S (1945) m ethod.
Phosphate-Solubilizing Microorganisms of a Lateritic Soil. II.
489
Table 1. Phosphate-solubilizing power in sucrose-rock phosphate broth and cellulose-decomposing ability of the test microorganisms OrganIdentified as isms Coded as
P - solubilized in /lg/15 mg insoluble P/O.15 g sucrose c-onsumed
Growth in broth medium
H 2S producing ability
Cellulosedecomposing power
Incubation in days Mean 10
7
19.5
24.0
21.8
Mucoid
9.0
9.0
9.0
Turbid
15.5
17.0
16.3
Asperqillu» sp.
18.5
26.0
22.3
A. niger van Tieghem
21.0
23.5
22.3
LCS 4
Bacillus [irmus (B-7647) B. circulans + B. subtilis (B-7648) Streptomyces sp.
LCF 3 LCF4
LCB l LCB 9
- nil,
Surface pellicle Surface mycelial pad Surface mycelial pad
+ +
+ positive
Results Among the isolates studied, LOF a, an unreported species of Aspergillus, and LOF 4 , Aspergillus niger van Tieghem, solubilized the highest amount of phosphorus from rock phosphate, followed by LOBI> Bacdiu« firmus (B-7647), L084 , Streptomyces sp., and LOB 9 , a mixed culture of B. circulans and B. subtilis (B-7648), while only the cited aspergilli possessed the ability of decomposing cellulose, and that to a considerably high degree (Table 1). None of the isolates could produce H 28. The sequence presented above and in the subsequent text will denote the order. As regards the growth type in sucrose-rock phosphate broth, LOB! gave mucoid, LOB g turbid, the streptomycete L08 4 surface pellicle, and the aspergilli gave surface mycelial pad. Partial sterilization by autoclaving eliminated more than 99 % of total bacteria and fungi and more than 98 %of the total actinomycetes, while completely eliminating the phosphate solubilizers (Table 2). The available phosphorus content of non-sterile and partially sterilized soils was essentially the same like in the untreated and uninoculated control soils (Table 3). Presence of manurial amendment uniformly decreased the content under partial sterilization, irrespective of any inoculation. This was also true for the non-sterile soil, excepting that inoculated with LOF a, Aspergillus sp., and L08 4 , Streptomyces sp., in presence of FYM and inoculated with LOB g, a mixed culture of B. circulans and B. subtilis (B-7648), in presence of FYM + RP. On the other hand, inoculation of LOBI> B. [irmu» (B-7647), decreased and LOF a, LOF 4, A. niger van Tieghem, and LOB 9 increased available phosphorus content of the partially sterilized soil under control untreated series, while L084 had no effect. In the non-sterile soil under this series, inoculation of LOF 4 and L08 4 increased and that of LOBI' LOB 9, and LOF 3 decreased the content. The overall effect of inoculation, considering all the manurial treatment and state of soil, in terms of partially sterilized and non-sterilized together, was significant in increasing available phosphorus content of the soil. In this respect, increases due to inoculation of LOF 4 and LOF a were significant as compared to control, followed by those due to LCFa, LCB 9 , L084 , and LCB I. The overall effect of manurial treatments was significantly depressing in terms of available phosphorus content of the soil, the most being all with rice straw alone.
490
S. BANIK and B. K. DEY
Table 2. Total and phosphate-solubilizing microbial population of the soil before and after partial
sterilization') Number (x 104 ) per g dry soil
Partial Sterilization
Bacteria
Streptomycetes
Before After
460.0 3.0
170.0 3.0
140.0 1.0
770.0 7.0
80.0 0
4.5 0
224.5 0
Fungi
Total
Phosphate-solubilizing microorganisms: P source: Ca 3(P04 h Before After
140.0 0
1) Soil autoclaved in thin layers at 15 Ib steam pressure for 30 minutes on two consecutive days.
Table 3. Available phosphorus content of soil on inoculation of some isolated microorganisms after 15 days of incubation Organisms inoculated
Soil
Available P content in kgjha (average of three sets) Manurial treatments Control
Control uninoculated
RP2)
FYM
FYM +RP
RS
RS Mean Mean for 2 sets +RP
II
10.29 10.08
5.46 3.45
5.46 7.56
6.38 7.72
8.44 3.32
6.22 6.16
7.04 6.38
6.71
LCB l 3)
I II
9.79 9.16
4.83 4.83
6.53 8.41
7.74 8.49
9.21 0
6.17 6.56
7.37 6.24
6.80
LCB 9
I II
11.16 9.84
5.13 4.41
6.09 9.58
7.56 11.13
8.31 0
5.59 5.96
7.30 6.82
7.06
LCS 4
I II
10.29 11.09
5.25 4.56
6.89 11.47
6.45 9.34
8.42 0
3.86 5.92
6.86 7.06
6.96
LCF 3
I II
12.14 Hl.05
5.75 5.81
6.69 12.39
9.00 8.07
9.17 0
6.47 6.55
8.20 7.14
7.67
LCF4
I II
12.00 12.06
4.92 11.18
6.85 8.67
7.59 9.58
9.30 0
5.86 7.08
7.68 8.09
7.88
Mean
I II
10.94 10.38
5.22 5.70
6.41 9.68
7.45 9.05
8.80 0.55
5.69 6.37
7.40 6.95
7.17
10.66
5.46
8.04
8.25
4.67
6.03
7.17
Mean for two sets
II)
For manurial treatment (M) Organism (0) Interaction Soil (S) X (M) two M at the same S Interaction (S X M) two S at the same or different M
CD at 5% 1.33 0.66 1.88 2.61
at 1% 1.94 0.92 2.74 3.61
Available P of initial soil: 12.5 kgjha
1) I: Soil autoelaved in thin layers (see Table 2), 2) II: Non-eterilized soil, and 3) see text.
Phosphate-Solubilizing Microorganisms of a Lateritic Soil. II.
491
Discussion Growth type in broth had an implication on solubilization of rock phosphate by the bacterial isolates, as LCB l , Baoillus lirmus (B-7647), producing mucoid growth, was found to be a better solubilizer than LCB 9 , a mixed culture of B. circulans and B. subtilis (B-7648), producing turbid growth (Table 1). Unlike earlier reports (SWABY and SPERBER 1959), the highest solubilization by the fungi, LCF" Aspergillus niger van Tieghem and LCFa, an unreported species of Aspergillus, was reached with sporulation. This may be attributed to the greater demand of phosphorus during sporulation (GILMAN 1957) and hence more solubilization, which was also true for LCS 4 , a Streptomyoes sp. Better survival of the general streptomycetes in the soil after partial sterilization by autoclaving, as compared to other microflora (Table 2), may be ascribed to their higher heat resistance (ALEXANDER 1977). However, this was not corroborated with the observation with the phosphate-solubilizing ones which were found to be very much sensitive to the process. Partial sterilization of soil had virtually no significant effect on available phosphorus content of the soil after 15 days of incubation, irrespective of any inoculation (Table 3). This indicates that the inoculated organisms were efficient enough to withstand and overcome the competition of the soil microorganisms. The universal decrease in the available phosphorus content after the said incubation may be ascribed to microbial immobilization of phosphorus. Contrary to the expectation (BHATTACHARYA and DEBNATH 1977), manurial treatment, in general, helped in net immobilization rather than solubilization of the element. The soil acidity (pH 5.4) might have restricted the proliferation of the nutritional symbionts which was presumably reflected on the metabolism of the phosphate solubilizers, resulting in a net immobilization of the element. Another reason might be that the inoculants at the first stage of growth produced soluble phosphorus in excess, which obviously improved the environment favourably for flourishing the immobilizers. Rectification of soil pH by liming and intensifying organic manuring (SARKAR 1973) would possibly reverse the process. On the other hand, a significant average increase in the available phosphorus content of the soil, due to inoculation of LCF4 , Aspergillus niger van Tieghem and LCFa, an unreported Aspergillus species, over the control may be attributed to their ability of utilizing cellulosic materials as energy substrate (Table 1), which was especially true with FYM, but surprisingly not with rice straw manure. The reason remained obscure. Possibly FYM had an additional indirect effect, as would be discussed later. The average accumulation of a relatively higher amount of available phosphorus in the partially sterilized soil, as expected, may be explained in the light of less immobilization, due to lesser number of microorganisms present after the process and higher solubilization by inoculated organisms in absence of sizeable competition. However, the higher accumulation of available phosphorus by Aspergillus sp., LCFa, Streptomyces sp., LCS" and LCB 9 , a mixed culture of B. circulans and B. subtilis in presence of FYM, the latter with rock phosphate supplementation, in non-sterile soil points out that these organisms were benefitted, in terms of phosphate solubilization, by the association of soil microorganisms, most probably by those elaborating growth-promoting substances, incited in presence of FYM. A drastic fall in available phosphorus content of the normal soil, due to application of straw manuring, supports intense immobilization. The situation was improved with the supplementation of rock phosphate. From the foregoing discussion it may be concluded that in soil, at least in absence of vegetation, the manures induced net immobilization of phosphorus, and in presence of them even the scant population survived after partial sterilization of the soil and
492
S. B ANIK a nd B. K. DEY, Phosphate. Solu bilizing Micro orga n ism s of a L a t eritic Soil. II.
were act ive in t he pr ocess when the conditions were fav ourabl e. Th e sit uation could only be revers ed with inocula tion of appropriate ph osphat e solubilizers . Ac kno w le dgeme nt The authors t hank t he Cou n cil of Sc ient ific and I ndust ri al R ese a r ch , I n d ia , for p ro viding ne cessary finan cial fa cilities.
References ALEXANDER, J\I. : I ntroduct ion to So il Microb iolog y. J ohn W iley & Sons In e., New Y ork an d Londo n 197 7. ALLEN, O. N . : Expe r ime n ts in Soi l Ba ct eriolog y. Burgess Publish in g Co., "Min neapo lis, Minnesot a 1957. B ANIK, S" an d DEY, B. K.: Phosphat e -solubilizing m icroorganism s of a lat er it ic soil. 1. Solubilization of in organic phosphat es and p rodu ct ion of organic aci ds by mi croorgan isms , isol ated in sucrose ca lc ium phosphate agar plates. Zbl. B akt. II 136 (19 81),478 -486. B HATTACHARYA, B. K., and DEBNATH, N . C. : Stu d ies on phosphate- sol ubilizing efficien cy of som e organic man ures. Indian Agric. J. 21 (19 77), 207 - 210. BRAY, R . H" an d KURTZ, L. '1'.: Deter mina t ion of total, organic an d availa ble forms of phosphorus in soils. Soil Sci. 59 (1945), 39 -45. F ARDEAU, .1 . C., an d GUIR.AURD, G. : Mo bilit y o f phosphorus in a so il rec e iv in g farm yard manure for t hi rt y five years. Co m p t. R end, 57 (19 71), 1598- 1605 (Soils an d F e rt, 35 [1972], 372 6). GILMAN, .1. C. : A man ua l of so il fungi . 2nd ed . Iowa St ate College Press 1958. GOLEBIOWSKA, J. : A me t hod of in vest ig at ion of t he mi crobial population playing a part in t he tmnsformation of phos pho ru s com po u nds in t he so il. Acta J\l icrobioL Polan. 5 (1956), 125 -127 (Soi ls a nd F ert. 20 [1 95. ], 94). H AYMAN, D. S. : P hosph or us cyding by soil mi croorganisms a nd plant roots. In : Soil Micr obiology (W ALKER, N ., ed . ), But t e r wort hs, Lo ndon 1975, 67 - 92. P IKOVSKAIA, R . I,: Mobilizat ion of phosphorus in soil in con ne ct ion with v it al a ct iv ity of some microb ial species . :Mik rob iologiy a 17 (1948 ), 362 - 370 . R UDRA, P .: Ch a racter ist ics a nd genesis of some of t he lateri tic soi ls of W est B engal leading to t he ir classification a n d land us e plann ing. P h .D . T he sis, Calcutta Un iv., Calcutta , India (1975). SARKAR, A. K. : Effect o f vario us p ho sphatic fertilizers in com b in at ion with lim e and ni trogen on the fertilit y im pro vement of lateritic soils . In d ian Agr ic. 7 (19 73), 1 - 6. SALLE, A. J . : L a b ora t o ry m anual on principles o f ba ct eri olog y. 6thed. McG raw Hill B ook Co. In c., New York 1967. SINKlIA, }I. K . : Effect o f co m pos tin g so il wit h straw on mobilit y and u pt ake by pla nts o f di fficultly soluble soi l p hosp hates. Iz v. Timiryazev. sel-k hoz. Ak ad. 6 (1970), 222- 225 (Soils and F ert , 34 [1 971],1 897). SWABY, H. ,J. , an d SPERBER, .J. 1. : P hosphate -dissolv ing mi croo rg an ism s in t he r h izosphe re of legu mes. N utrition of legumes. (HALLSWORTH, E. D., ed.) But t er wort h s. L ondon 1959, 289 294. Author 's addresses : D r. S. B ANTK, U n ive rsit y Colleg e of Ag ri cult ure, Microbiological L aborator y, Ca lc ut t a University, calcutt a 700 01 9; a nd Dr. B. K. DE Y, Dep ar t m ent of Agricu lt u ral Chemist ry and Soil Scien ce, F a cult y of Agric ultu re , B idhan Chandra K r ishi Vi sw a Vid ya laya, K al yani, N adia, Pin 741 235, W est Bengal, Indi a.
[University College o f Agriculture, Microbiological Laboratory, Calcutta University, Calcutta, India]
Phosphate-Solubilizing Microorganisms of a Lateritic Soil D. Effect of Some Tricalcium Phosphate-Solubilizing Microorganisms on Available Phosphorus Content of the Soil") S.
BANIK
and B. K.
DEY
Summary Five effic ien t phosphate-solubilizing microo rganisms isolated from a lateritic soil (Typic Ochragualf), L OBI' Bacillue [irmu« (B-7647), LOB" a mixed culture of Bacillus circulan8 and B. 8ubtili8 (B-7648), LCS., a Streptomgce» sp., LCF3 , an unreported species of A8pergillu8, and LOF., A. niger van Ti eghem, were inoculated in the said soil with rock phosphate (RP), farm yard manure (FYM), FYM + HP, rice straw (RS), and RS + RP in a flask culture experiment to study their role in impro ving the available phosphorus status of the soil under naturals non-sterile, and partially sberitized con d it ion s. The aspergilli L OF 3 and LOF. and the Bacillus LCB I showed their efficiency in solubilizing rock phosphate in broth cult ure. The aspergilli were also efficient cell ulose decomposers. Partial sterilization by autoclaving of the soil had no significant effec t on available phosphorus con ten t of the soil, irrespecti ve of a n y ino culation. Inoculation of L OF. and LCS. increased the available phosphorus con ten t of the unmanured soil. Application of manures, es pecia lly that of ri ce straw, lowe red the con ten t, wh er eas inoculation of LOF3 and LCS. with FYM a nd that of LOB, with FYM + HP in cr e ased the a vailable phosphorus con tent of t he soil. Howe ver, a s ignif ica n t effe ct of inoculation was obta ined only with L OF 3 an d L CFt in the respective cases m entioned.
Zusammenfassung Eine m L ate r-it (t yp. Ochragualf) wurde Rohphosphat, Stalldung und Reisst r oh e in zeln und in K ombination zugesetzt. Von flinf a us di ese m Boden isolierten Mikroorganismen erh oht en Aspergillu 8 n ige r und S treptomgces s pec. den Ge halt an verfiigbarem Phosphat des unbehandelten Bod en s. AHe Zu satze a u 13er Rohphospha t verringerten das ver fiigba re Phosphat, insbesondere R eisst ro h . Signifikante Erhohungen e rgab di e Beimpfung mit A. nig er und A spe rqillu» spe c. Irn einzelnen wirkte Beimpfung mit d en beiden Aspergillen in del' Variante Stalldung - ni cht steril und Beimpfung mi t eine r Mischkultur von B acillue ci rcul an8 und B. 8ubti li 8 in del' Variante Stalldung + R eisstroh - ni cht steril deutlich er h ohe n d au f den Gehalt an verftlgbarem Phosphat.
Among the different groups of phosphate-solubilizing microorganisms present in soil (GOLEBIOWSKA 1956), the performance of the group dissolving tricalcium phosphate in excess of the requirement appears to be ecologically and agronomically very important under Indian conditions, as most of the soil and natural phosphorus are lock ed in tricalcium phosphate. Carbonaceous materials play are crucial role when biochemical activity of these chemo-organotrophs is concerned . Supplementation of natural rock phosphate with organic manures enhances the proliferation of the phosphate solub ilizers and, as a consequence, intensifies solubilization of insoluble inor ganic phosphates in the soil (SI NKHA 1970; FARDEAU and G UIRAURD 1971). Bacteri1) P art of t he Ph.D. T hesis, su b m it ted b y the fir st author t o the U n ive rs ity of Ca lc ut t a in 1979.
488
S. B ANIK a nd B. K. D EY
zation with t he resp onsible microflora accent ua tes t he process (HAYMAN 1975). The present work was undertak en in order to obtain information regarding t he accumulation of a vailable phosphate in a lat eri ti c soil as influenced by t he inoculation of some efficient phosphat e-solubilizing microorganisms, isolated fr om the soil, in absence and presence of organic manures with or without rock phosphate under partially sterilized a nd non- sterilized na tural cond it ions.
Materials and Methods Source of isolates T he cult ures used in t his st ud y were selecte d from those isolat ed from a lateritic soil (Typic Ochragu alf, R UDRA 197 5) in P IKOVSKA IA'S (1948) suc rose ca lc ium phos phate aga r pl ates, showing hi gh phosphat e-solubilizing ab ility (BANIK and DEY 1981). T hese were L CBI' B acillus [irmu« (B -764 7); L CB~, a mixed cult u re of Bacillus circulans an d B. subtilis (B· 764 8); L CS 4, Strep tomyces sp. ; L CF s' an unreported sp ecies of Asperqillu»; an d LCF4 • Aspergill us niger van Tieghem (BANIK a n d DE Y 1981). The cultures we re maintaine d on the said PIK OVSKAIA' S (194 8) medium. The cu lt ures were tested fo r t hei r a b il ity of solubilizing rock phosphate an d de composing cellulo se, as ro ck phosphate and organic mat ter, cont ain ing appreciable amount of cell ul osic materials, we re included in the treatments wit h so il in t he su bseq uent experiment, and H 2S-producing ability is one of t he mean s of liberating phosphate from F eP0 4 present in t he soil.
Solubilizati on of rock phosphate Phosp h a t e-di ssol ving abili t y of t he isolates fr o m rock phosphat e was deter mine d in m od ified PIKOVSKAIA'S (1948) broth , containi ng 20 g ro ck phosphate, h a ving a n eq uivalent a mount of phosphor us as in 5.0 g CaS (P 0 4)2' T he p rocedures were t he sa me as foll owed b y B ANIK a n d DEY (198 1).
Decomposition of cellulose Ability to decompose cellulo se, i.e., to utilize cell ul ose as sole so urce of ca rbo n an d energy, was t est ed in Du bos' me d ium (ALLEN 195 7), us ing filter pape r strips in triplicate. The method of ino culat ion of t he isol ates was sim ilar to t hat follo wed by BANIK and DEY (198 1), T he tubes were incub ated at 30 ± 1 °C for one m onth. T he extent of deterioration of t he fil t er pa per str ips indicat ed the cell ulo se-decom posing abifit.y.
Production of H 2S T he ability for p roducing H 2S was t ested b y bl ackening of lea t acetat e-filter p a per str ips as descr ibe d by SALLE (196 7).
Avail abl e pho sphorus content of t he soil as affected b y inoculation of the isolates F or est im at ing the abilit y of t he isolates for bringing una vailable phosphorus to an avail able state in soil, 10.0 g lots of the sa me soil (BANIK a nd DEY 1981) h arbouring the isolates, were taken in 100 ml conical flasks with the foll owing treatments: (a) Untreated cont ro l ; (b) Rock phosphate (RP) , 30 kg p . ha - I ; (c) Farm y ar d manu re (FYM), 40 kg N· h a- I ; (d ) F YM, 40 kg N + RP, 30 kg p . h a - I ; (e) Rice straw (RS) , 40 kg N· h a - I ; and (f) RS, 40 k g N + RP, 30 kg p. ha: ", The mo isture le vel was kept at 100 % wa t er h olding cap ac it y, since the su bs eque nt experiment was proposed to be done on ri ce rhi zosphere under flooded con dit ion . F or eac h t reat me n t 36 fl asks we re ke pt. Out of t hese , 18 flasks we re autoclaved at 15 1b stea m pressure (121 ec ) for 30 m inutes on two con sec ut ive days, i.e. , so-called pa rtial steril ization , with a view t o co m pa re t he activity of the ino culated isola tes unde r a minimal microbia l co m pet it ion in soil. Partially steril ized a nd n on-st e ril e flask s we re inoculat ed with t he isolates in triplicate peI' manurial treatment. The proce dure of in ocul at ion was t he same as t hat ad o pte d b y BANIK an d DEY (198 1). The fl ask s were incu bated at 30 ± 1 °C. T he moisture level was maintaine d at 100 % wat e r h old in g ca pacity b y aseptica lly a dd ing sterile d istilled water e ve r y alternate d a y. Aft er 15 d a ys of incubat ion, t he available p ho sp horus of t h e fla sk content was est im at ed , following BRAY a n d K URTZ' S (1945) m ethod.
Phosphate-Solubilizing Microorganisms of a Lateritic Soil. II.
489
Table 1. Phosphate-solubilizing power in sucrose-rock phosphate broth and cellulose-decomposing ability of the test microorganisms OrganIdentified as isms Coded as
P - solubilized in /lg/15 mg insoluble P/O.15 g sucrose c-onsumed
Growth in broth medium
H 2S producing ability
Cellulosedecomposing power
Incubation in days Mean 10
7
19.5
24.0
21.8
Mucoid
9.0
9.0
9.0
Turbid
15.5
17.0
16.3
Asperqillu» sp.
18.5
26.0
22.3
A. niger van Tieghem
21.0
23.5
22.3
LCS 4
Bacillus [irmus (B-7647) B. circulans + B. subtilis (B-7648) Streptomyces sp.
LCF 3 LCF4
LCB l LCB 9
- nil,
Surface pellicle Surface mycelial pad Surface mycelial pad
+ +
+ positive
Results Among the isolates studied, LOF a, an unreported species of Aspergillus, and LOF 4 , Aspergillus niger van Tieghem, solubilized the highest amount of phosphorus from rock phosphate, followed by LOBI> Bacdiu« firmus (B-7647), L084 , Streptomyces sp., and LOB 9 , a mixed culture of B. circulans and B. subtilis (B-7648), while only the cited aspergilli possessed the ability of decomposing cellulose, and that to a considerably high degree (Table 1). None of the isolates could produce H 28. The sequence presented above and in the subsequent text will denote the order. As regards the growth type in sucrose-rock phosphate broth, LOB! gave mucoid, LOB g turbid, the streptomycete L08 4 surface pellicle, and the aspergilli gave surface mycelial pad. Partial sterilization by autoclaving eliminated more than 99 % of total bacteria and fungi and more than 98 %of the total actinomycetes, while completely eliminating the phosphate solubilizers (Table 2). The available phosphorus content of non-sterile and partially sterilized soils was essentially the same like in the untreated and uninoculated control soils (Table 3). Presence of manurial amendment uniformly decreased the content under partial sterilization, irrespective of any inoculation. This was also true for the non-sterile soil, excepting that inoculated with LOF a, Aspergillus sp., and L08 4 , Streptomyces sp., in presence of FYM and inoculated with LOB g, a mixed culture of B. circulans and B. subtilis (B-7648), in presence of FYM + RP. On the other hand, inoculation of LOBI> B. [irmu» (B-7647), decreased and LOF a, LOF 4, A. niger van Tieghem, and LOB 9 increased available phosphorus content of the partially sterilized soil under control untreated series, while L084 had no effect. In the non-sterile soil under this series, inoculation of LOF 4 and L08 4 increased and that of LOBI' LOB 9, and LOF 3 decreased the content. The overall effect of inoculation, considering all the manurial treatment and state of soil, in terms of partially sterilized and non-sterilized together, was significant in increasing available phosphorus content of the soil. In this respect, increases due to inoculation of LOF 4 and LOF a were significant as compared to control, followed by those due to LCFa, LCB 9 , L084 , and LCB I. The overall effect of manurial treatments was significantly depressing in terms of available phosphorus content of the soil, the most being all with rice straw alone.
490
S. BANIK and B. K. DEY
Table 2. Total and phosphate-solubilizing microbial population of the soil before and after partial
sterilization') Number (x 104 ) per g dry soil
Partial Sterilization
Bacteria
Streptomycetes
Before After
460.0 3.0
170.0 3.0
140.0 1.0
770.0 7.0
80.0 0
4.5 0
224.5 0
Fungi
Total
Phosphate-solubilizing microorganisms: P source: Ca 3(P04 h Before After
140.0 0
1) Soil autoclaved in thin layers at 15 Ib steam pressure for 30 minutes on two consecutive days.
Table 3. Available phosphorus content of soil on inoculation of some isolated microorganisms after 15 days of incubation Organisms inoculated
Soil
Available P content in kgjha (average of three sets) Manurial treatments Control
Control uninoculated
RP2)
FYM
FYM +RP
RS
RS Mean Mean for 2 sets +RP
II
10.29 10.08
5.46 3.45
5.46 7.56
6.38 7.72
8.44 3.32
6.22 6.16
7.04 6.38
6.71
LCB l 3)
I II
9.79 9.16
4.83 4.83
6.53 8.41
7.74 8.49
9.21 0
6.17 6.56
7.37 6.24
6.80
LCB 9
I II
11.16 9.84
5.13 4.41
6.09 9.58
7.56 11.13
8.31 0
5.59 5.96
7.30 6.82
7.06
LCS 4
I II
10.29 11.09
5.25 4.56
6.89 11.47
6.45 9.34
8.42 0
3.86 5.92
6.86 7.06
6.96
LCF 3
I II
12.14 Hl.05
5.75 5.81
6.69 12.39
9.00 8.07
9.17 0
6.47 6.55
8.20 7.14
7.67
LCF4
I II
12.00 12.06
4.92 11.18
6.85 8.67
7.59 9.58
9.30 0
5.86 7.08
7.68 8.09
7.88
Mean
I II
10.94 10.38
5.22 5.70
6.41 9.68
7.45 9.05
8.80 0.55
5.69 6.37
7.40 6.95
7.17
10.66
5.46
8.04
8.25
4.67
6.03
7.17
Mean for two sets
II)
For manurial treatment (M) Organism (0) Interaction Soil (S) X (M) two M at the same S Interaction (S X M) two S at the same or different M
CD at 5% 1.33 0.66 1.88 2.61
at 1% 1.94 0.92 2.74 3.61
Available P of initial soil: 12.5 kgjha
1) I: Soil autoelaved in thin layers (see Table 2), 2) II: Non-eterilized soil, and 3) see text.
Phosphate-Solubilizing Microorganisms of a Lateritic Soil. II.
491
Discussion Growth type in broth had an implication on solubilization of rock phosphate by the bacterial isolates, as LCB l , Baoillus lirmus (B-7647), producing mucoid growth, was found to be a better solubilizer than LCB 9 , a mixed culture of B. circulans and B. subtilis (B-7648), producing turbid growth (Table 1). Unlike earlier reports (SWABY and SPERBER 1959), the highest solubilization by the fungi, LCF" Aspergillus niger van Tieghem and LCFa, an unreported species of Aspergillus, was reached with sporulation. This may be attributed to the greater demand of phosphorus during sporulation (GILMAN 1957) and hence more solubilization, which was also true for LCS 4 , a Streptomyoes sp. Better survival of the general streptomycetes in the soil after partial sterilization by autoclaving, as compared to other microflora (Table 2), may be ascribed to their higher heat resistance (ALEXANDER 1977). However, this was not corroborated with the observation with the phosphate-solubilizing ones which were found to be very much sensitive to the process. Partial sterilization of soil had virtually no significant effect on available phosphorus content of the soil after 15 days of incubation, irrespective of any inoculation (Table 3). This indicates that the inoculated organisms were efficient enough to withstand and overcome the competition of the soil microorganisms. The universal decrease in the available phosphorus content after the said incubation may be ascribed to microbial immobilization of phosphorus. Contrary to the expectation (BHATTACHARYA and DEBNATH 1977), manurial treatment, in general, helped in net immobilization rather than solubilization of the element. The soil acidity (pH 5.4) might have restricted the proliferation of the nutritional symbionts which was presumably reflected on the metabolism of the phosphate solubilizers, resulting in a net immobilization of the element. Another reason might be that the inoculants at the first stage of growth produced soluble phosphorus in excess, which obviously improved the environment favourably for flourishing the immobilizers. Rectification of soil pH by liming and intensifying organic manuring (SARKAR 1973) would possibly reverse the process. On the other hand, a significant average increase in the available phosphorus content of the soil, due to inoculation of LCF4 , Aspergillus niger van Tieghem and LCFa, an unreported Aspergillus species, over the control may be attributed to their ability of utilizing cellulosic materials as energy substrate (Table 1), which was especially true with FYM, but surprisingly not with rice straw manure. The reason remained obscure. Possibly FYM had an additional indirect effect, as would be discussed later. The average accumulation of a relatively higher amount of available phosphorus in the partially sterilized soil, as expected, may be explained in the light of less immobilization, due to lesser number of microorganisms present after the process and higher solubilization by inoculated organisms in absence of sizeable competition. However, the higher accumulation of available phosphorus by Aspergillus sp., LCFa, Streptomyces sp., LCS" and LCB 9 , a mixed culture of B. circulans and B. subtilis in presence of FYM, the latter with rock phosphate supplementation, in non-sterile soil points out that these organisms were benefitted, in terms of phosphate solubilization, by the association of soil microorganisms, most probably by those elaborating growth-promoting substances, incited in presence of FYM. A drastic fall in available phosphorus content of the normal soil, due to application of straw manuring, supports intense immobilization. The situation was improved with the supplementation of rock phosphate. From the foregoing discussion it may be concluded that in soil, at least in absence of vegetation, the manures induced net immobilization of phosphorus, and in presence of them even the scant population survived after partial sterilization of the soil and
492
S. B ANIK a nd B. K. DEY, Phosphate. Solu bilizing Micro orga n ism s of a L a t eritic Soil. II.
were act ive in t he pr ocess when the conditions were fav ourabl e. Th e sit uation could only be revers ed with inocula tion of appropriate ph osphat e solubilizers . Ac kno w le dgeme nt The authors t hank t he Cou n cil of Sc ient ific and I ndust ri al R ese a r ch , I n d ia , for p ro viding ne cessary finan cial fa cilities.
References ALEXANDER, J\I. : I ntroduct ion to So il Microb iolog y. J ohn W iley & Sons In e., New Y ork an d Londo n 197 7. ALLEN, O. N . : Expe r ime n ts in Soi l Ba ct eriolog y. Burgess Publish in g Co., "Min neapo lis, Minnesot a 1957. B ANIK, S" an d DEY, B. K.: Phosphat e -solubilizing m icroorganism s of a lat er it ic soil. 1. Solubilization of in organic phosphat es and p rodu ct ion of organic aci ds by mi croorgan isms , isol ated in sucrose ca lc ium phosphate agar plates. Zbl. B akt. II 136 (19 81),478 -486. B HATTACHARYA, B. K., and DEBNATH, N . C. : Stu d ies on phosphate- sol ubilizing efficien cy of som e organic man ures. Indian Agric. J. 21 (19 77), 207 - 210. BRAY, R . H" an d KURTZ, L. '1'.: Deter mina t ion of total, organic an d availa ble forms of phosphorus in soils. Soil Sci. 59 (1945), 39 -45. F ARDEAU, .1 . C., an d GUIR.AURD, G. : Mo bilit y o f phosphorus in a so il rec e iv in g farm yard manure for t hi rt y five years. Co m p t. R end, 57 (19 71), 1598- 1605 (Soils an d F e rt, 35 [1972], 372 6). GILMAN, .1. C. : A man ua l of so il fungi . 2nd ed . Iowa St ate College Press 1958. GOLEBIOWSKA, J. : A me t hod of in vest ig at ion of t he mi crobial population playing a part in t he tmnsformation of phos pho ru s com po u nds in t he so il. Acta J\l icrobioL Polan. 5 (1956), 125 -127 (Soi ls a nd F ert. 20 [1 95. ], 94). H AYMAN, D. S. : P hosph or us cyding by soil mi croorganisms a nd plant roots. In : Soil Micr obiology (W ALKER, N ., ed . ), But t e r wort hs, Lo ndon 1975, 67 - 92. P IKOVSKAIA, R . I,: Mobilizat ion of phosphorus in soil in con ne ct ion with v it al a ct iv ity of some microb ial species . :Mik rob iologiy a 17 (1948 ), 362 - 370 . R UDRA, P .: Ch a racter ist ics a nd genesis of some of t he lateri tic soi ls of W est B engal leading to t he ir classification a n d land us e plann ing. P h .D . T he sis, Calcutta Un iv., Calcutta , India (1975). SARKAR, A. K. : Effect o f vario us p ho sphatic fertilizers in com b in at ion with lim e and ni trogen on the fertilit y im pro vement of lateritic soils . In d ian Agr ic. 7 (19 73), 1 - 6. SALLE, A. J . : L a b ora t o ry m anual on principles o f ba ct eri olog y. 6thed. McG raw Hill B ook Co. In c., New York 1967. SINKlIA, }I. K . : Effect o f co m pos tin g so il wit h straw on mobilit y and u pt ake by pla nts o f di fficultly soluble soi l p hosp hates. Iz v. Timiryazev. sel-k hoz. Ak ad. 6 (1970), 222- 225 (Soils and F ert , 34 [1 971],1 897). SWABY, H. ,J. , an d SPERBER, .J. 1. : P hosphate -dissolv ing mi croo rg an ism s in t he r h izosphe re of legu mes. N utrition of legumes. (HALLSWORTH, E. D., ed.) But t er wort h s. L ondon 1959, 289 294. Author 's addresses : D r. S. B ANTK, U n ive rsit y Colleg e of Ag ri cult ure, Microbiological L aborator y, Ca lc ut t a University, calcutt a 700 01 9; a nd Dr. B. K. DE Y, Dep ar t m ent of Agricu lt u ral Chemist ry and Soil Scien ce, F a cult y of Agric ultu re , B idhan Chandra K r ishi Vi sw a Vid ya laya, K al yani, N adia, Pin 741 235, W est Bengal, Indi a.