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A better technique for quantitative isolations of microfungi during biodegradation of agricultural wastes
International Biodeterioration 25 (1989) 57-64
A Better Technique for Quantitative Isolations of Microfungi During Biodegradation of Agricultural Wastes
A. P. Garg & B a b i t a B h a t n a g a r Department of Botany, Meerut University. Meerut - 250 005, India
ABSTRACT By using different time intervals for making different serial suspensions of almost equal concentration of spores/hyphae, a better technique for quantitative isolations of microfungi during the biodegradation of agricultural wastes has been developed and described. A comparison of microfungal populations obtained by this proposed 'serial washing technique' and the widely used dilution plate technique has been made. The technique may also be standardised for the quantitative isolations of microfungi from the phylloplane. It is expected that the technique will have wide application for quantitative isolation of microorganismsfrom various deteriorating~degrading materials in nature.
INTRODUCTION Since the recognition of the importance of fungi as decomposers in a food chain, several qualitative and quantitative isolation techniques for microfungi from leaves and leaf-litter of various plant species have been proposed (Preece & Dickinson, 1971; Dickinson & Pugh, 1974; Dickinson & Preece, 1976; Blakeman, 1981). For quantitative isolations the dilution plate technique has been widely used by microbial ecologists. Its several disadvantages have earlier been pointed out by Dickinson (1971) and Garg (1980). The use of different surface areas of leaves/leaf57 International Biodeterioration 0265-3036/89/$03.50© 1989 Elsevier Science Publishers Ltd, England. Printed in Great Britain.
A. P. Garg,BabitaBhatnagar
58
litter with different amounts of water at different suspension times and various serial washings for making a pooled undiluted suspension have given variable results. The use of 1 : 10 and 1 : 100 dilutions have further complicated the procedure of standard calculations. The authors have devised a better standard technique for quantitative isolations of fungal populations during the decomposition of agricultural waste materials.
MATERIALS A N D M E T H O D S Five plant species, maize (Zea mays), triticale (Triticosecale hexaploide), guar (Cyamopsis tetragonoloba), wheat (Triticum aestivum) and sorghum (Sorghum vulgate)were selected for the standardisation of the technique. The crops were sown in 5 m X 5 m plots in their respective seasons under the r e c o m m e n d e d agronomical practices.
Collection of samples The dead leaves at the end of the growing season were collected aseptically and placed separately in fresh sterile nylon bags mesh size 500pm and left in the fields. For wheat, triticale and guar whole leaves were placed in the bags while for maize and sorghum the leaves were cut into two to three pieces. For each species four bags were placed on the soil surface at four different sites in the field. Samples often leaves/leaf parts were collected at r a n d o m from the bags each time. Except for maize, samples were collected at intervals of 30 days until they fragmented. Maize samples were collected at intervals of 20 days until all litter fragmented into the soil. A total of 100 squares 5 m m × 5 mm, 10 from each of the 10 leaves/leaf parts were cut at r a n d o m aseptically with a sterile parallel razor. Fifty were processed for the 'serial washing technique' and fifty for the dilution plate technique as follows.
Serial washing suspensions Fifty leaf squares were placed in a 500 ml sterile Erlenmeyer flask containing 100 ml of double distilled sterile water and shaken for 30 s at 100 rpm. The suspension minus the leaf squares was poured into a 250 ml sterile flask A. The leaf squares were shaken again for 1, 2, 5, 10, 15 and 25 min each time with fresh 100 ml double distilled sterile water, and
Serial washing techniquefor isolation of microfungi
59
p o u r e d separately into fresh sterile 250 ml flasks a n d labelled B, C, D, E, F a n d G respectively. The flasks were covered with sterile a l u m i n i u m foil t h r o u g h o u t the procedure. Aseptic conditions were m a i n t a i n e d during the investigation. Five replicates of 1 ml aliquots of each serial washing were m a d e with 20 ml sterile cool m o l t e n P D A containing streptomycin (to avoid bacterial growth). Sterile 10 c m diameter Petri dishes were used during the whole investigation. The plates were incubated at 25 _+ 1°C for 7-10 days a n d the colonies identified, c o u n t e d a n d recorded. The calculations were m a d e as follows: Total no. of colonies of species 'X" in all S.W. X P o p u l a t i o n of species 'X" = no. of S.W. X a m o u n t of water / c m 2 leaf surface area No. of replicates X total surface area where, S.W. = serial washings Since the procedures were standardised calculated: N u m b e r of serial washings = a m o u n t of water = n u m b e r of replicates =
the following factor was 7; 100 ml; 5;
total surface area (both surfaces) = 0.5 X 0.5 X 2 X 50 cm 2 = 25 cm 2 Thus,
the factor -
7 X 100 5 X 25 5.6
The p o p u l a t i o n of species 'X' / c m 2 leaf surface area
Total no. of colonies of species 'X" in all S.W. X 5-6
Dilution plate I (DP I) The r e m a i n i n g 95 ml of suspension of each of the above serial washings were p o o l e d together a n d treated as u n d i l u t e d suspension a n d 1 : 10 a n d 1 : 100 dilutions made. Ten replicates of 1 ml aliquots for each of these suspensions were m a d e a n d studied similarly to the serial washing technique.
Calculations Total populations were calculated for undiluted, 1 : 1 0 a n d 1:100 dilutions separately each time taking the original u n d i l u t e d suspension
A. P Gang Babita Bhatnagar
60
obtained from 25 cm 2 leaf surface area to be 700 ml, by using the following formula: P o p u l a t i o n of species 'X" =
Total no. of colonies of species 'X' in all 10 replicates x 700 X dilution factor
/ c m 2 leaf surface area
No. of replicates × total surface area
Total fungal populations on five litter stages of maize are shown in Table 1 as obtained from undiluted, 1 : 10 a n d 1 : 100 dilutions.
Dilution plate II (DP II) This m e t h o d was followed by using 2 ml o f w a t e r / c m 2 leaf surface area, as used by most previous investigators. The r e m a i n i n g 50 leaf squares were s h a k e n in 250 ml sterile flask six times with fresh 50 ml double distilled sterile water a n d pooled in a 500 ml sterile flask. The 300 ml suspension was treated as u n d i l u t e d suspension from which 1 : 1 0 a n d 1:100 dilutions were prepared. Ten replicates of 1 ml aliquots for each of the u n d i l u t e d a n d diluted suspensions were m a d e with 20 ml cool molten P D A + streptomycin, incubated a n d studied as for the serial washing technique. Calculations for the fungal populations were m a d e according to the D P I technique. Results for u n d i l u t e d a n d each of the diluted suspensions are shown in Table 1 as the total fungal populations per cm 2 leaf surface area. RESULTS AND DISCUSSION Total fungal populations per cm 2 of leaf surface area (both surfaces) colonising various litter stages of Zea mays obtained by serial washing a n d the two dilution plate techniques are shown in Table 1. With the serial washing technique a c o n t i n u o u s decrease in total fungal populations from L2 to Ls stage was observed. However, with D P I a n d II techniques no general pattern emerged. Undiluted, 1 : 10 a n d 1 : 100 dilutions in D P I a n d II techniques showed greater differences in fungal populations. Averaging these figures to assess the density of fungal populations on the leaf surface does not seem to be convincing. Microbial ecologists use different dilutions to avoid competition between species o n agar plates. In our serial washing technique, we have avoided competition by using serial suspensions of lower fungal density to give not more t h a n 20 colonies per plate. A higher total n u m b e r of species by the serial washing technique t h a n either of the D P m e t h o d s (Table 2) indicates the greater efficiency of the former technique.
Serial washing techniquefor isolation of microfungi
61
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Serial washing techniquefor isolation of microfungi
TABLE 3 Average Number of Colonies per Plate Isolated by Serial Washing Technique from Five Plant Species (Average of Five Replicates) Serial washing
A B C D E F G
Suspension time
30 sec 1 min 2min 5min 10min 15min 25min
Average number of colonies Maize
Triticale
Guar
Wheat
Sorghum
14 14 19 12 15 14 9
16 15 18 17 16 15 10
19 18 19 21 15 13 10
16 12 15 16 14 12 7
17 17 15 14 12 12 6
Various c o m b i n a t i o n s of time intervals between the successive washings were tried to obtain an average of 15 + 5 colonies o n agar plates in each of the serial washings. T h e results are shown in Table 3 as average n u m b e r of colonies developing on the agar plates. The colonies were neither overlapping n o r crowded a n d could be c o u n t e d a n d identified easily. After the seventh washing the n u m b e r of colonies fell to 0-3, indicating that most o f the fungal hyphae/spores were detached by this time. Therefore, serial washing for 30 s, 1, 2, 5, 10, 15 a n d 25 m i n each time with 100 ml double distilled sterile water for 25 cm 2leaf surface area can be used for quantitative isolations of microfungi from the leaflitter of herbaceous plant species. However, it is suggested that the procedure should be standardised for each application. The populations obtained by our p r o p o s e d serial washing technique are representative of 5% of the total s u s p e n s i o n a n d are therefore more accurate than the dilution plate method. The populations obtained by leaf-imprint techniques a n d direct observation on various plant species also support the range obtained by serial washing technique (Sharma et al, 1974: Garg, 1980; Garg & Sharma, 1984, 1985). Garg (1988) has successfully standardised this technique for the quantitative isolation of microfungi from p h y l l o p l a n e of herbaceous plant species by using 2, 5, 10, 20, 35 a n d 50 m i n as the time interval during successive washings. However, it is suggested that this serial washing technique needs further detailed investigations u n d e r different climatic conditions on other types of substrates. ACKNOWLEDGEMENTS A.P.G. wishes to t h a n k the University Grants C o m m i s s i o n , New Delhi for financial support during this investigation. A.P.G. also wishes to
64
A. P Garg Babita Bhatnagar
t h a n k the D e u t s c h e r A k a d e m i s c h e r Austauschdienst, Bonn, Federal Republic of G e r m a n y for the travel grant to present this work in the VII International Biodeterioration S y m p o s i u m held at Cambridge, 6-11 September 1987. T h e authors are indebted to Professor Dr J. MOiler, Institut for Medizinische Mikrobiologie u n d Hygiene, der Universit~t Freiburg, FRG, for his valuable suggestions a n d discussions, a n d to Professor Dr R. N. Smith, The Hatfield Polytechnic, a n d Dr J. Mills, Sheffield City Polytechnic, UK, for editing the manuscript.
REFERENCES Blakeman, J. P. (ed.) (1981).MicrobialEcology of the Phylloplane. Academic Press, London and New York. Dickinson, C. H. (1971). Cultural studies on leaf saprophytes. In Ecology of Leaf Surface Microorganisms, ed. T. F. Preece & C. H. Dickinson. Academic Press, London and New York, pp. 129-37. Dickinson, C. H. & Preece, T. F. (eds) (1976). Microorganisms of Aerial Plant Surfaces. Academic Press, London and New York. Dickinson, C. H. & Pugh, G. J. F. (eds) (1974). Biology of Plant Liner Decomposition, 2 vols. Academic Press, London and New York. Garg, A. P. (1980). Studies on microfungi associated with living and dead leaves of Triticosecale and Cyamopsis. PhD Thesis, Meerut University, India. Garg, A. P. (1988). Comparison ofphylloplane microbes assessment techniques and proposal of a new serial washing technique for quantitative isolations of microfungi from leaf surfaces. International Conference on Research in Plant Sciences and its Relevance to the Future. New Delhi, India, 7-11 March 1988 p. 116. Garg, A. P. & Sharma, P. D. (1984). Ecology of phylloplane and litter fungi of triticale. Nordic Journal of Botany, 4, 707-15. Garg, A. P. & Sharma, P. D. (1985). Ecology of phylloplane and leaf-litter fungi of Cyamopsis tetragonoloba (L.) Taub. Revue d'Ecologie et de Biologie du Sol, 22, 35-55. Preece, T. F. & Dickinson, C. H. (eds) (1971). Ecology of Leaf Surface Microorganisms. Academic Press, London and New York. Sharma, IC R., Behra, N. & Mukerji, K. G. (1974). A comparison of three techniques for assessment of phylloplane microbes. Transactions of the Mycological Society of Japan, 15, 223-33.
A Better Technique for Quantitative Isolations of Microfungi During Biodegradation of Agricultural Wastes
A. P. Garg & B a b i t a B h a t n a g a r Department of Botany, Meerut University. Meerut - 250 005, India
ABSTRACT By using different time intervals for making different serial suspensions of almost equal concentration of spores/hyphae, a better technique for quantitative isolations of microfungi during the biodegradation of agricultural wastes has been developed and described. A comparison of microfungal populations obtained by this proposed 'serial washing technique' and the widely used dilution plate technique has been made. The technique may also be standardised for the quantitative isolations of microfungi from the phylloplane. It is expected that the technique will have wide application for quantitative isolation of microorganismsfrom various deteriorating~degrading materials in nature.
INTRODUCTION Since the recognition of the importance of fungi as decomposers in a food chain, several qualitative and quantitative isolation techniques for microfungi from leaves and leaf-litter of various plant species have been proposed (Preece & Dickinson, 1971; Dickinson & Pugh, 1974; Dickinson & Preece, 1976; Blakeman, 1981). For quantitative isolations the dilution plate technique has been widely used by microbial ecologists. Its several disadvantages have earlier been pointed out by Dickinson (1971) and Garg (1980). The use of different surface areas of leaves/leaf57 International Biodeterioration 0265-3036/89/$03.50© 1989 Elsevier Science Publishers Ltd, England. Printed in Great Britain.
A. P. Garg,BabitaBhatnagar
58
litter with different amounts of water at different suspension times and various serial washings for making a pooled undiluted suspension have given variable results. The use of 1 : 10 and 1 : 100 dilutions have further complicated the procedure of standard calculations. The authors have devised a better standard technique for quantitative isolations of fungal populations during the decomposition of agricultural waste materials.
MATERIALS A N D M E T H O D S Five plant species, maize (Zea mays), triticale (Triticosecale hexaploide), guar (Cyamopsis tetragonoloba), wheat (Triticum aestivum) and sorghum (Sorghum vulgate)were selected for the standardisation of the technique. The crops were sown in 5 m X 5 m plots in their respective seasons under the r e c o m m e n d e d agronomical practices.
Collection of samples The dead leaves at the end of the growing season were collected aseptically and placed separately in fresh sterile nylon bags mesh size 500pm and left in the fields. For wheat, triticale and guar whole leaves were placed in the bags while for maize and sorghum the leaves were cut into two to three pieces. For each species four bags were placed on the soil surface at four different sites in the field. Samples often leaves/leaf parts were collected at r a n d o m from the bags each time. Except for maize, samples were collected at intervals of 30 days until they fragmented. Maize samples were collected at intervals of 20 days until all litter fragmented into the soil. A total of 100 squares 5 m m × 5 mm, 10 from each of the 10 leaves/leaf parts were cut at r a n d o m aseptically with a sterile parallel razor. Fifty were processed for the 'serial washing technique' and fifty for the dilution plate technique as follows.
Serial washing suspensions Fifty leaf squares were placed in a 500 ml sterile Erlenmeyer flask containing 100 ml of double distilled sterile water and shaken for 30 s at 100 rpm. The suspension minus the leaf squares was poured into a 250 ml sterile flask A. The leaf squares were shaken again for 1, 2, 5, 10, 15 and 25 min each time with fresh 100 ml double distilled sterile water, and
Serial washing techniquefor isolation of microfungi
59
p o u r e d separately into fresh sterile 250 ml flasks a n d labelled B, C, D, E, F a n d G respectively. The flasks were covered with sterile a l u m i n i u m foil t h r o u g h o u t the procedure. Aseptic conditions were m a i n t a i n e d during the investigation. Five replicates of 1 ml aliquots of each serial washing were m a d e with 20 ml sterile cool m o l t e n P D A containing streptomycin (to avoid bacterial growth). Sterile 10 c m diameter Petri dishes were used during the whole investigation. The plates were incubated at 25 _+ 1°C for 7-10 days a n d the colonies identified, c o u n t e d a n d recorded. The calculations were m a d e as follows: Total no. of colonies of species 'X" in all S.W. X P o p u l a t i o n of species 'X" = no. of S.W. X a m o u n t of water / c m 2 leaf surface area No. of replicates X total surface area where, S.W. = serial washings Since the procedures were standardised calculated: N u m b e r of serial washings = a m o u n t of water = n u m b e r of replicates =
the following factor was 7; 100 ml; 5;
total surface area (both surfaces) = 0.5 X 0.5 X 2 X 50 cm 2 = 25 cm 2 Thus,
the factor -
7 X 100 5 X 25 5.6
The p o p u l a t i o n of species 'X' / c m 2 leaf surface area
Total no. of colonies of species 'X" in all S.W. X 5-6
Dilution plate I (DP I) The r e m a i n i n g 95 ml of suspension of each of the above serial washings were p o o l e d together a n d treated as u n d i l u t e d suspension a n d 1 : 10 a n d 1 : 100 dilutions made. Ten replicates of 1 ml aliquots for each of these suspensions were m a d e a n d studied similarly to the serial washing technique.
Calculations Total populations were calculated for undiluted, 1 : 1 0 a n d 1:100 dilutions separately each time taking the original u n d i l u t e d suspension
A. P Gang Babita Bhatnagar
60
obtained from 25 cm 2 leaf surface area to be 700 ml, by using the following formula: P o p u l a t i o n of species 'X" =
Total no. of colonies of species 'X' in all 10 replicates x 700 X dilution factor
/ c m 2 leaf surface area
No. of replicates × total surface area
Total fungal populations on five litter stages of maize are shown in Table 1 as obtained from undiluted, 1 : 10 a n d 1 : 100 dilutions.
Dilution plate II (DP II) This m e t h o d was followed by using 2 ml o f w a t e r / c m 2 leaf surface area, as used by most previous investigators. The r e m a i n i n g 50 leaf squares were s h a k e n in 250 ml sterile flask six times with fresh 50 ml double distilled sterile water a n d pooled in a 500 ml sterile flask. The 300 ml suspension was treated as u n d i l u t e d suspension from which 1 : 1 0 a n d 1:100 dilutions were prepared. Ten replicates of 1 ml aliquots for each of the u n d i l u t e d a n d diluted suspensions were m a d e with 20 ml cool molten P D A + streptomycin, incubated a n d studied as for the serial washing technique. Calculations for the fungal populations were m a d e according to the D P I technique. Results for u n d i l u t e d a n d each of the diluted suspensions are shown in Table 1 as the total fungal populations per cm 2 leaf surface area. RESULTS AND DISCUSSION Total fungal populations per cm 2 of leaf surface area (both surfaces) colonising various litter stages of Zea mays obtained by serial washing a n d the two dilution plate techniques are shown in Table 1. With the serial washing technique a c o n t i n u o u s decrease in total fungal populations from L2 to Ls stage was observed. However, with D P I a n d II techniques no general pattern emerged. Undiluted, 1 : 10 a n d 1 : 100 dilutions in D P I a n d II techniques showed greater differences in fungal populations. Averaging these figures to assess the density of fungal populations on the leaf surface does not seem to be convincing. Microbial ecologists use different dilutions to avoid competition between species o n agar plates. In our serial washing technique, we have avoided competition by using serial suspensions of lower fungal density to give not more t h a n 20 colonies per plate. A higher total n u m b e r of species by the serial washing technique t h a n either of the D P m e t h o d s (Table 2) indicates the greater efficiency of the former technique.
Serial washing techniquefor isolation of microfungi
61
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Serial washing techniquefor isolation of microfungi
TABLE 3 Average Number of Colonies per Plate Isolated by Serial Washing Technique from Five Plant Species (Average of Five Replicates) Serial washing
A B C D E F G
Suspension time
30 sec 1 min 2min 5min 10min 15min 25min
Average number of colonies Maize
Triticale
Guar
Wheat
Sorghum
14 14 19 12 15 14 9
16 15 18 17 16 15 10
19 18 19 21 15 13 10
16 12 15 16 14 12 7
17 17 15 14 12 12 6
Various c o m b i n a t i o n s of time intervals between the successive washings were tried to obtain an average of 15 + 5 colonies o n agar plates in each of the serial washings. T h e results are shown in Table 3 as average n u m b e r of colonies developing on the agar plates. The colonies were neither overlapping n o r crowded a n d could be c o u n t e d a n d identified easily. After the seventh washing the n u m b e r of colonies fell to 0-3, indicating that most o f the fungal hyphae/spores were detached by this time. Therefore, serial washing for 30 s, 1, 2, 5, 10, 15 a n d 25 m i n each time with 100 ml double distilled sterile water for 25 cm 2leaf surface area can be used for quantitative isolations of microfungi from the leaflitter of herbaceous plant species. However, it is suggested that the procedure should be standardised for each application. The populations obtained by our p r o p o s e d serial washing technique are representative of 5% of the total s u s p e n s i o n a n d are therefore more accurate than the dilution plate method. The populations obtained by leaf-imprint techniques a n d direct observation on various plant species also support the range obtained by serial washing technique (Sharma et al, 1974: Garg, 1980; Garg & Sharma, 1984, 1985). Garg (1988) has successfully standardised this technique for the quantitative isolation of microfungi from p h y l l o p l a n e of herbaceous plant species by using 2, 5, 10, 20, 35 a n d 50 m i n as the time interval during successive washings. However, it is suggested that this serial washing technique needs further detailed investigations u n d e r different climatic conditions on other types of substrates. ACKNOWLEDGEMENTS A.P.G. wishes to t h a n k the University Grants C o m m i s s i o n , New Delhi for financial support during this investigation. A.P.G. also wishes to
64
A. P Garg Babita Bhatnagar
t h a n k the D e u t s c h e r A k a d e m i s c h e r Austauschdienst, Bonn, Federal Republic of G e r m a n y for the travel grant to present this work in the VII International Biodeterioration S y m p o s i u m held at Cambridge, 6-11 September 1987. T h e authors are indebted to Professor Dr J. MOiler, Institut for Medizinische Mikrobiologie u n d Hygiene, der Universit~t Freiburg, FRG, for his valuable suggestions a n d discussions, a n d to Professor Dr R. N. Smith, The Hatfield Polytechnic, a n d Dr J. Mills, Sheffield City Polytechnic, UK, for editing the manuscript.
REFERENCES Blakeman, J. P. (ed.) (1981).MicrobialEcology of the Phylloplane. Academic Press, London and New York. Dickinson, C. H. (1971). Cultural studies on leaf saprophytes. In Ecology of Leaf Surface Microorganisms, ed. T. F. Preece & C. H. Dickinson. Academic Press, London and New York, pp. 129-37. Dickinson, C. H. & Preece, T. F. (eds) (1976). Microorganisms of Aerial Plant Surfaces. Academic Press, London and New York. Dickinson, C. H. & Pugh, G. J. F. (eds) (1974). Biology of Plant Liner Decomposition, 2 vols. Academic Press, London and New York. Garg, A. P. (1980). Studies on microfungi associated with living and dead leaves of Triticosecale and Cyamopsis. PhD Thesis, Meerut University, India. Garg, A. P. (1988). Comparison ofphylloplane microbes assessment techniques and proposal of a new serial washing technique for quantitative isolations of microfungi from leaf surfaces. International Conference on Research in Plant Sciences and its Relevance to the Future. New Delhi, India, 7-11 March 1988 p. 116. Garg, A. P. & Sharma, P. D. (1984). Ecology of phylloplane and litter fungi of triticale. Nordic Journal of Botany, 4, 707-15. Garg, A. P. & Sharma, P. D. (1985). Ecology of phylloplane and leaf-litter fungi of Cyamopsis tetragonoloba (L.) Taub. Revue d'Ecologie et de Biologie du Sol, 22, 35-55. Preece, T. F. & Dickinson, C. H. (eds) (1971). Ecology of Leaf Surface Microorganisms. Academic Press, London and New York. Sharma, IC R., Behra, N. & Mukerji, K. G. (1974). A comparison of three techniques for assessment of phylloplane microbes. Transactions of the Mycological Society of Japan, 15, 223-33.