The effect of soil moisture content on the cellulolytic mycoflora of soil amended with cellulosic remains

Microbiol. Res. (1996) 151, 301- 308

Microbiological Research © Gustav Fischer Verlag Jena

The effect of soil moisture content on the cellulolytic mycoflora of soil amended with cellulosic remains R. A. M. Badran 1 , A. Abdel-Rahiem 2 1

2

Botany Department, Faculty of Science, Kena Botany Department, Faculty of Science, Sohag

Accepted: October 15, 1995

Abstract The fungal popUlation of soil amended with four types of cellulosic remains under different levels of water content was recorded. 40 - 80% soil water content was the best level for fungal multiplication. There was no specific fungal flora isolated from any of the three cellulosic remains but some fungi grew noticeably better on some cellulose types than on others. The gross total count of fungi was the highest in soil amended with sugar cane straw at 60% water content but the least was in soil mixed with broad bean straw at 100% water content. Aspergillus, Penicillium, Chaetomium, Cladosporium, Emerciella nidulans, Fusarium and Stachybotrys were the most dominant fungi. Sugar cane straw was the most hospitable cellulosic material for soil funig. 60% water content was more suitable for the cellulolytic activity of test fungi but protein content was the highest in between 60 - 80% of soil water content. Key words: Dry soil - cellulosic remains -

content - cellulolytic fungi.

soil water

Introduction Cellulose is by far the most prominent organic compound in nature and its decomposition by soil microorganisms has received a considerable attention because of its significance in the biological cycle of carbon (Griffiths and Jones 1963; Park and McKee 1978; Moustafa and Sharkas 1982; EI-Sharouny et. al. 1989; Moubasher and Mazen 1991 and Amalia et. al. 1993).

Corresponding author: R. A. M. Badran

Water content is therefore an important factor influencing microbial activity in the natural environment. Changes in soil moisture content have resulted in rapid shifts of microbial biomass (Bottner 1985; Amalia et al. 1993) and soil drying-rewetting cycles have a significant influence on microbial activity (Kieft et al. 1987; Taylor and Parkinson 1988). Many investigators tested the cellulolytic activities of fungi in pure cultures (Walsh and Stewart 1969; Malik and Eggins 1970; Moharam 1984). The ability of the organism to utilize the native cellulose depends to its ability to produce cellulase (Norkrans 1963; Flannigan 1970; El-Sharouny et al. 1984). In Egypt, few and limited studies have been made on the cellulolytic activity of soil fungi (Moharam 1984; Shaban 1986; Moubasher and Mazen 1991), but we have no available knowledge on the role of soil water content on cellulose decomposition. Hence our investigation was designed to study the population and composition of cellulose decomposing soil fungi under different levels of water which are expected to change profoundly as a response to deep changes in soil and climatic factors.

Materials and methods Treatment of soil. A lot of 20 kilos of sandy soils collected from the East Bank of Kena Governorate was dried at room temperature (35 - 40 DC) for four weeks. The soil was dispensed into clean plastic bags (20 bags, each contains one kilo). These bags were classified into 5 groups; 3 bags of each group were mixed individually with 5 g of sterilized powder of the Microbiol. Res. 151 (1996) 3

301

selected cellulosic matter and the rest bag served as control. Sterilized distilled water was used to adjust the required different levels of soil moisture content (as mentioned in Garrett 1981). The bags were tied and incubated at 28°C for 15 days.

Isolation and identification of soil fungi. The dilution plate method as described by Johnson et al. (1959) was used for estimation of soil fungi. Czapek's agar medium in which glucose was replaced by cellulose (20 gil) was employed. Rose bengal (1/30000) + streptomycin (20 mg/ml) as bacteriostatic agents (Martin 1950) were added to the isolation medium. 5 plates were used for each sample and incubated at 28°C for 15 days. The developed colonies were identified and calculated per g dry soil. The following references were used for the identification of fungal genera and species: Raper and Thom (1949), Raper and Fennel (1965) and Domsch et al. (1980). Screening test for cellulolytic activity. For exocellulase (Cd: All the isolated fungi were grown on Eggins and Pugh's liquid medium (1962) for 7days at 28°C. One ml of the clear supernatent of each fungus was pipetted in wells made in the solid medium of Eggins and Pugh's. After 24 h incubation, the plates were flooded with saturated solution of zinc iodide. The clear zones which appeared were measured and used as criterion for cellulolytic activity. For endocellulase (Cx): The same fungal isolates were grown on the same medium of Eggins and Pugh's medium in which insoluble cellulose was replaced by carboxymethylcellulose and the above method was employed. Determination of cellulolytic activities in cellulosic remains-treated soil. The screening test proved that Aspergillus fumigatus, Chaetomium globosum, Cladosporium herbarum, Penicillium chrysogenum, Stachybotrys chartarum and Trichoderma viride were the most potents for cellulase production. This recommends studying their cellulolytic activities in soil. Each 100 g of soil was mixed throughly with 5 g of sugar cane straw or broad bean straw or wheat straw individually. The moisture content of these soil samples readjusted using sterilized water to get 20%, 40%, 60%, 80% and 100% water holding capacities. The soil samples were put in clean sterilized plastic bags and each set was inoculated individually by each of test fungi. These bags were tied and incubated at 28°C for 30 days. At the end of the incubation period, the reducigng sugars were determined using Nelson method (1944) and also the protein content of each soil sample was estimated according to Lowry et al. (1951). 302

Microbiol. Res. 151 (1996) 3

Results The results of table (1) showed that the number of fungal genera and species were affected markedly by the soil water content and the selected cellulosic matter. The total population increased regularly by increasing the water content and the highest numbers were detected in between 40 - 80% water content then decreased thereafter. 3540.7 colonies/ g dry soil (the highest number) were detected in soil amended with sugar cane straw at 60% water content. The least number (123 colonies/g dry soil) was estimated in soil amended with broad bean straw at 100% water content. However according to the effect of soil water content on fungal population, our results can be classified to the following categories (Table 1): A-In case of soil amended with sugar cane straw, the fungal counts are 495.5, 1562.4, 3540.7, 2093.5 and 153 colonies/g dry soil at 20%, 40%,60%,80% and 100% of water content respectively. B-On the other hand, and in soil incorporated with wheat straw, 265, 841.5, 2009.7, 728.2 and 179 colonies/g dry soil at 20%, 40%, 60%, 80% and 100% of water content respectively. C-In case of broad bean-incorporated soil, 193, 600.5,1143.5,615 and 123 colonies/g dry soil at 20%, 40%,60%,80% and 100% of water content respectively. It is worthmentioning that sugar cane straw is the most hospitable matter at most levels of water. Penicillium (8 species) was the leader genus and was recorded all through the experiment with the highest count (892.5 colonies/g dry soil) at 60% water content. P. chrysogenum followed by P. oxalicum and P. corylophilum were the most common species. Aspergillus was among the most common genera. Seven species of it were recorded of which A.flavus, A. fumigatus and A. niger were common. Most of Aspergillus species showed their heighest population in 40 - 80% of soil water content. On the other hand, A. versicolor represented 122 colonies/g dry soil in sugar cane-incorporated soil at 80% water content. The following fungal species including Aremonium strictum, Chaetomium globosum, Cladosporium c/adosporioidies, C. herbarum, Emerciella nidulans, Fusarium moniliforme, Rhizopus stolonifer, Stachybotrys chartarum, Trichoderma viride and 1hchothecium roseum were encountered in high frequencies in this experiment especially in between 40 - 80% of soil water content. Generally the fungal population was markedly decreased in soil of 100% water content (Table 1). In contrast, Trichurus spira lis emerged only in high count (117 colonies) in soil mixed with wheat straw at 100% water content.

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Alternaria alternata Nees & Keissler Acremonium A. implicatum Gilman & Abb. Gams A. strictum W. Gams Aspergillus A.flavus Link A.fumigatus Fresenius A. niger Van Tieghem A. ochraceus Wilhelm A. sydowii (Bain. Sart) Thorn & Church A. tamarii Kita A. versicolor (Yuill) Trab. Beauveria bassiana Bals. & Yuill Botryotrichum piluliferum Sacco & March Chaetomium C. globosum Kin. Fr. C. olivaceum Cooke & Ellis Cladosporium C. cladosporioides Fres. De Vries C. herbarum (Pers.) Link: Fr. Cochliobolus specifera Nelson Emerciella nidulans (Eidam) Yuill Fusarium E. moniliforme Sheldon E. solani (Mart.) Sacco Gliocladium roseum Bain Humicola grisea Traaen

Fungal genera and species

Water content of soil

6

5

1

3

8 5

5

17.5

2

25 25

6

24

24

15

3

87

5

79

5

1

8

101 14

2

2

2

11 9

22

11.5

20.5 9

6

13.5

5 5

11.5

5

2

3

8 3

4 1

5

2

2

2

5

1

3

12 9

14

1

3

7 4

218

4 3 1

17

5

5

80 75

239

4

13

4

36 23

85

9

86

195

5

19

41 22

92

7

34.5

46.5 12

31 27 4

101 85 16

6

17

16

101 15

22

12

9

9

8

52 44

29

13

11

14 3

4

10 6

5

4

2 1

3 13

35.5

2

39 5 3

8

2

10

2

Cont.

15 89 15 13.5

6

21

45

B. st

17

217

9

209

9

3

1

14

3

15

9

5

5 1

3 3

2 3

114 15 69

75 4 45

15 2

15.5 2

62.5

9

3

1

29

1 5

15 247 15 17

18 241.9 17.9 35

956 95.5 44.5 39 10 14 2 14 11 9

5

20

2

20

14

9 3

54 13

5

5

22

18

13 4

14

9 2

5

10

40%

7

2

15

3

Cont.

B. st

105

45

95

508 413

271

62

312

409 97

397 299 98

88

15

39 55

14 114

7

65 310 47 34

76 11

3.2

79

9

37

129 92

93

17

94

138.7 44.7

248 191 57

115

87

15 38 52

44 293 59 42

61 17

2

3

21

78 57

102

15

19

46

27

4

79 75

96

14

11 27

3

4

2

2

9

11

3

2

1

3 2

2

15 13

7

1

1 2

4 1 2

52

99

9

23 14

92

29

108 79

2

15 13

53

45

122

79

19

5 2

5

244 12

14

2 2

9

4

19 113 43 15

9

2

7 3

2

31 12

3

4

13

14

23 9

63

17

16

33

2 2

44

89.5

4

13

11

4

7

2

15 13

25

13

25 12

4 4

17

3

2

11

2

1

3

121.5 16

3

3

2

4 4

2

2

1

3 2

4 4

2

1

1

2

2

2 2

10

2

2

3

12

15

12

15

27 12

5

2

4

2

8

4

4

2 2

3

14

5

19

2

6 2 4

1

1

2

2

5 3

3 3

B. st

Table 1. Fungal counts (per g dry soil) isolated from soil amended with three cellulosic materials at different levels of water content and recovered on cellulose-Czapek's medium at 28 °C after 15 days of incubation.

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5

5

495.5 265 26 24 17 15

320 42 25

2

43

24.5 7

11

14

111

3 39 4

15 14

15

5

2

5

69 29 5

211

14 2

9

64 3

115

9

2

67

5

29.7

14

53

59

5 14

2

17.5

141 72 44.5

27 12

38

7

11

214

1 17

7

4

126 95 19

83 7

! B. st

2093.5 728.2 615 35 29 24 22 19 16

44

315

137

9

295

78

3

11 19

120

2

49

11

18

7

19

11 144

13

93

331.5 193 25.5

39 39

2

53 15 5 2 1 9

45 2

w. st

80% Cont.! S. st !

35

24

7

125

11 16 2 2

57.5

248.5 84 78

94 12

I B. st

3540.7 2009.7 1143.5 591 42 39 34 40 25 24 20 24

44 93

78

121

68

92 75

54 89 74 3

2 44

454 97 94

5 44

w. st

58.5

11

17 39 219

892.5 345

I

60%

8

9

2

3

34

4 69

57

5 3 7 4

8

18

9

27

74 34 9 5 3

8 57

19 4

19 7 90 39 15

s. st

Cont.I

B. st

1562.4 841.5 600.5 546 30 42 36 34 21 19 18 25

3

19

155 40 24

94

12

3

68

57

9

9

14

2

11

5 17.5 9

14

67 15 13 9

3 13

w. st I

15 58

47

5

5

5

4 65

2 3

1

4

6

4

I

40%

214.5 94 44

7 19

14 7 30 9 1 3

s. st

Cont.I

2

193 24 14

5

17

56

6 2

3

19

3

4

6

12

52 23 7

4

45

15 19

3

2 3 2

8

9

24 4

9

2 24

52 15

5

25

4

2

2

9

4 1 2 3

6

21 5 1 2

2

s. st I w. st I B. st

20%

= Control; S. st = Sugar cane straw; w. st = Wheat straw; B. st = Broad bean straw.

Gross total count No. of species No. of genera

6

Mucor racemosus Fres. Myrothecium roridum Tode Penicillium P. chrysogenum Thorn P. corylophilum Diercks P. funiculosum Thorn P. janthinellum Biourge P. oxalicum Chur. & Thorn P. purpurogenum Stoll P. roqueforti Thorn P. rubrum Stoll Paecilomyces variotii (Thorn) Samson Papulospora sp. Rhizopus stolonifer (Ehrenb): Fr. Scolecobasidium variabile Barron & Busch Stachybotrys chartarum (Ehrenb.: Lindt) Hughes Talaromyces stipitatus Thorn & Benj Trichoderma viride Pers.: S. F. Gray Trichothecium roseum (Pers.) Link: Fr. Trichurus spiralis Hasselbring Verticillium lateritium Berk. 4

Cont.I

Fungal genera and species

Water content of soil

(Continued)

153 19 13

45

91

210 28 19

2

3

2

2

9

3

4

26 10

2

3

19

13

4

25

2

3

6 2

11

9 4

179 13 11

117

3

7

9

7

4 4

123 10 9

94

2

3

5

2

2

Cont. I s. st I w. st I B. st

100%

90 80 70 60

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C·

50

eIJ

40

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30

20 10 0

20%

80%

60°1c

40%

100%

90 80

70 c

60

V'

50

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40

30 20 10

o 20"10 Fig. 1

40%

60%

80%

100%

Water content of soil Microbiol. Res. 151 (1996) 3

305

70

60 ,.., oJ

50

:.I:

c·

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40

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0 0

30

....... ;lJ

E

Control A fum 9 tu Cglobo um C h rbarum • P chrysog num

20 10

S.char1arum

• T.vlrld

0 20%

40Vo

60%

80%

100%

Fig. 1. Effect of soil water content on the cellulolytic activity of some fungi inoculated in soil amended with (A) Sugar cane straw, (B) Broad bean straw, (C) wheat straw

The remaining fungal genera and species were less encountered (Table 1). It is to be mentioned that there was no basic differences in the composition of the fungal flora of cellulosic materials during all the experiment but some fungi were noticeably promoted on some selected cellulosic matter than others. Data of Fig. 1 illustrated that 60 - 80% of soil water content was the best level for cellulase activity by test fungi. The highest levels of protein content were obtained at 60 - 80% of soil water content, too (Fig. 2).

Discussion According to the data presented in table (1) we can conclude that sugar cane straw is the most hospitable matter for most of fungal species. All of these fungal species recorded in table (1) have been reported by many authors as cellulose decomposers (Reese and his co-workers, 1950, 1951; Walsh and Stewart 1969; Malik and Eggins 1970; EI-Nawawy et. al. 1984; Moubasher and Mazen 1991). Nancy Kokalis et. al. (1994) proved that fungal populations were positively increased in soil treated with pine park and Penicillium chrysogenum and Paecilomyces variotii were the dominant species. Penicillium (8 species), Aspergillus (7 species) were common in the first part of investigation. Pugh et. al. (1963) and Pugh (1964) listed Penicillium species among microbial species capable of utilizing cellu306

Microbiol. Res. 151 (1996) 3

lose. Besides, Raper and Fennel (1965) reported that all members of Aspergillus appeared to be capable of hydrolyzing cellulose. Generally the fungal population, their cellulolytic activities and protein contents were markedely decreased at soil of 100% water content. This may be due to the shortage of oxygen in these heavy irrigated soils. Garrett (1981) proved that carbon dioxide, especially in soil of high levels of water content and in micro sites of rapid respiration, may increase to a partial pressure at which it becomes fungistatic. Most of test fungi exhibited their highest cellulolytic activities at 60% water content (Fig. 1) and also the protein content were detected in the best levels at 60% water content in case of soil amended with sugar cane straw and wheat straw (Fig. 2) but at 80% water content in soil amended with broad bean straw. The wide variation of the Egyptian soil water content was the limiting factor on the fungal count and their composition. It seems that the buildup of fungal population at the site of study may clarify the role played by fungi in the biodegradation of cellulosic wastes in our soil which is subjected to deep changes of climatic factors. The final results of this breakdown of dead plant tissue is the liberation of essential mineral nutrients needed by higher plants in a soluble form which can be taken up from the soil by their roots. Finally and accordigng to the results showed above, we can advise that the biodegradation of the

(A)

.-o-

80

en

70

~

60 SO

o o

40

bO

-

30 20

1: L-~~~~==~~==~d§~~~~ 20%

40%

80%

60%

100%

(8)

70

~

on o

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60 50 40

30

20

1: L-~~~~~========~====~~ 20',4

40'10

80',4

60'"

100',4

(C)

80

~

bO

-o o

on

E

Fig. 2. Effect of soil water content on protein content of some fungi inoculated in soil amended with (A) Sugar cane straw, (B) Broad bean straw, (C) Wheat straw

70

60 50 40 30 20

10 o L-~~+=~==~~~~==~~==~~ 100% 20% 80% 40% 60% water content of soli

cellulosic wastes under suitable condition of irregation, especially sugar cane straw, which is the main crop in our Governorate, may play an important role to increase the fertility of our arid soils of East and West Desert in Egypt and the improvement of soil texture may be achieved. Acknowledgement The authors thank Prof. Dr. H. M. EI-Sharouny for his valuable help in discussing this work.

--Control -0-- A.fumigatus -- C.globosum - x- C.herbarum -- P.chrysogenum -<>- S.chartarum - 0 - T.viride

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Moubasher, A. H., Mazen, M. B. (1991): Assay of cellulolytic activity of cellulose-decomposing fungi isolated from Egyptian soil. J. Basic Microbiol. 31, 59-68. Moustafa, F., Sharkas, M. S. (1982): Fungi associated with cellulose decomposition in the tidal mud flats of Kuwait. Mycopathol. 78, 185 -190. Nelson, N. (1944): A photometric adaptation of the Somogyi method for the determination of glucose. J. BioI. Chern. 153,375-380. Norkrans, B. (1963): Degradation of cellulose. Ann. Rev. Phytopathol. 1, 325 - 350. Park, D., McKee, W. (1980): Cellulolytic Pythium as a component of River mycoflora Trans. Brit. Mycol. Soc. 71,251-259. Pugh, G. J. F. (1964): An investigation of soil borne cellulose-decomposing in Greece. Ann. Inst. Phytopathol. Banaki. 7, 19-27. Pugh, G. J. F., Morgan, J. G., Eggins, H. O. W. (1963): Studies of fungi in coastal soils. Trans. Brit. Mycol. Soc. 46, 565 - 571. Raper, K. B., Fennel, D. I. (1965): The genus Aspergillus. Williams and Wilkins, Baltimore, U.S.A. Raper, K. B., Thorn, C. (1949): A manual of the Penicillia, Williams and Wilkins, Baltimore, U.S.A. Reese, E. T., Dowiving, M. H. (1951): Activity of Aspergilli on cellulose, cellulose derivatives and wool. Mycologia 43, 16-23. Reese, E. T., Siu, R. G. H., Levinson, H. S. (1950): The biological degradation of soluble cellulose derivatives and its relationship to mechanism of cellulose hydrolysis. J. Bact. 59, 485-497. Shaban, M. M. Gehan (1986): Physiological and ecological studies in the genus Trichoderma in Egyptian soils. Ph. D. Thesis, Botany Dept. Fac. Sci. EI-Minya University, Egypt. Taylor, B. R., Parkinson, D. (1988): Does repeated wetting and drying accelerate decay of leaf litter? Soil BioI. Biochem. 20, 647 - 656. Walsh, J. M., Stewart, C. S. (1969): A simple method for the assay of cellulolytic activity of fungi. Intern. Biodeter. Bull. 5, 15-20.