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Competitive saprophytic colonization of pigeon-pea substrate by Fusarium udum in relation to environmental factors, chemical treatments and microbial antagonism
Soil Biol. Biochem. Vol. 15, No. 2, pp. 187-191, 1983 Printed in Great Britain. All rights reserved
0038-0717/83/020187-05$03.00/O Copyright C 1983 Pergamon Press Ltd
COMPETITIVE SAPROPHYTTC COLONIZATION OF PIGEON-PEA SUBSTRATE BY PUSARZUM UDUA4 IN RELATION TO ENVIRONMENTAL FACTORS, CHEMICAL TREATMENTS AND MICROBIAL ANTAGONISM BHARAT RAI and R. S. UPADHYAY Department
of Botany,
Banaras
Hindu
University,
Varanasi
221005,
India
(Accepted 20 July 1982)
Summary-The competitive saprophytic colonization (CSC) of pigeon-pea substrates by Fusurium udum was studied in relation to environmental factors, chemical treatments and microbial antagonism. CSC was best at 22”C, good at 3O”C, but at 40°C the substrate was vigorously colonized by Coprinus lagopus and Aspergillus niduluns which suppressed colonization by F. udum. High competitive saprophytic ability of F. udum was noted at pH 7 and 9 at soil moisture contents between 5 and 30%. At pH 4 and 5 F. udum was replaced by A.javus and A. nidulans. The CSC of F. udum decreased in the soil-sand inoculum mixture amended with the fungicides bavistin, dithane Z-78 and difolatan. The herbicides 2,4-D and machete also reduced CSC by F. udum whereas urea promoted it. Colonization of pigeon-pea substrate by F. udum was highly suppressed by antagonism from Penicillium citrinum, A. niger, Micromonospora globosa, A.Jlaous, A. terreus and Trichoderma Gride when these were used in inoculum mixtures with F. udum or when substrates had already been colonized by them. Otherwise F. udum was shown to have a high competitive saprophytic ability.
INTRODUCTION Fusarium
udum Butler is a soil-inhabiting pathogenic root-infecting fungus which causes wilt disease of pigeon-pea (Cujanus cujan (L.) Millsp.). The extent of ability of a soil-borne plant pathogen to survive saprophytically in the absence of its host depends on its competitive saprophytic ability; this may be assessed by competitive saprophytic colonization of substrate units buried in graded series of inocu1um:soil dilutions (Garrett, 1950). Competitive saprophytic ability in relation to biological control of soil-borne diseases has been discussed by Garrett (1965) and Park (1965). We have investigated the competitive saprophytic colonization of pigeon-pea substrate by F. udum in relation to moisture, pH, temperature, chemical treatments and microbial anatgonism.
MATERIALS AND METHODS
The Cambridge method (Garrett, 1963) was followed. Pure inoculum of the test pathogen was prepared in 500 ml conical flasks each containing 200 g sterilized acid-washed sand + 3% maize meal. Each flask was inoculated with three blocks (10 mm dia) of agar culture of F. udum. Moisure in the sand was maintained at 25’j/, and its pH was adjusted to 6.5. The flasks were incubated at 25 k 2°C for 10 days. The flasks were shaken periodically so as to maintain a homogeneous continuum of propagules. The inoculum-sand mixture of F. udum, prepared in this way, was mixed with field soil in different proportions (w/w), i.e. lOO:O, 98:2, 90:10, 50:50, 10:90, 2:98 187
and 0: 100; the first and last serving as respective controls for inoculum and field soil. The soil sample was collected from a pigeon-pea field situated near Broacha Hostel, Banaras Hindu University Campus. The soil contained a high resident population of F. udum due to wilt disease of pigeon-pea in the field (0.61-1.40 x lo3 g-’ dry soil; Upadhyay and Rai, 1982) and this was confirmed by repeated isolation of its colonies on Martin’s agar medium (Upadhyay, 1979). Moisture in the inoculum-soil mixture was maintained at 25 + 3% except where mentioned otherwise. The substrate used included roots and branches of stem from healthy pigeon-pea plants collected from the field at harvest. Fifty pieces (2cm length and 0.2-0.5 cm dia) of sterilized substrate were buried in 250 ml conical flasks containing 1oog of inoculum-soil mixture. The flasks were held at 25 f 1°C for 4 weeks and then the substrate units were taken out and washed 10-12 times in sterile distilled water. The washed pieces were surface sterilized with 0.1% HgC12 (in 10% alcohol) for 1 min then rinsed in sterile distilled water. The pieces were then blotted between the folds of sterile blotting papers and were transferred to Petri dishes (5 pieces/dish) containing 20 ml Czapek-Dox agar medium. Each treatment was replicated three times. All Petri dishes were held at 22 & 2°C for 6 days and the percentage colonization of root/shoot substrates by the test fungus was recorded. From graphs of per cent colonization against inoculum dilution the api.e. the perproximate C,, value was calculated, centage of non-sterile soil permitting colonization of 50% of the substrate units by F. udum (Garrett, 1975).
BHAKAT RAI
188
Non-biological
and R. S.
,fbctors studied
The effects of the following were studied in triplicate sets of all soil--inocuIum mixtures: ---Soil moisture at 5, 15, 25 and 30% moisture {oven-dry basis) maintained with sterile distilled water. -Soil reaction adjusted to pH 4, 5, 7 and 9 by dilute acid or alkali. --Soil temperature by incubation at 15 & 2,22 + 2, 30 2 2 and 40 k 2°C. -The three fungicides dithane Z-78 (75% zinc ethylene ~j.~-dithio~arbamate and 25% inert ingredients, Indofil Chemicals Ltd, Bombay), difolatan (80% cis,N-( I ,1,2,2-tetrachoroethyl) (thio) 4-cyclohexane I, dicarboxymide, Rallies India Ltd, Bombay) and carbamoylbenzimidazole, bavistin (7%~ Methoxy BASF India Ltd. Bombay). 0.1,O.S and 1.O?/, concentrations of the fungicides were at 20% moisture level. Herbicides. 0.1, 0.5 and 1.0% concentration of 2,4-D (2,4-dichlorophenoxy acetic acid, 80% active ingredient) and machete (2-chloro-2’,6’-diethyl-N(butoxymethyl) acetanilide, 50% active ingradient). Urcu. 0.1. 0.5 and I .O:/, concentrations.
Aspergiks fiarus Link ex Fries, A. niger van Tieghem, A. terreus Thorn, Coprinus lugopus Fr., Pupulasporu sp., Penicillium citrinum Thorn, Rhizoctoniu solani Kiihn, Trichoderma viride Pers ex Gray, Micromonospara glohosa Krass. and one yellow bacterium were used. Pure inocula of these test lni~rooganisms were prepared separately in sterilized sand + 3”i;, maize meal in conical flasks. Four hundred sterile root segments (2cm length and 0.2-0.5cm dia) of pigeon-pea were buried in each flask and were held at 25 & 1°C for 20 days. The root segments were then removed, washed thoroughly and transferred to 250 ml conical flasks containing 100 g different dilutions of sand-inoculum (100, 9X,90, 50, 10 and 2’;d diluted with field soil) of F. udum. The flasks were then held at 25 + 1°C for 4 weeks. Thereafter, the root segments were washed several times with distilled water, surface sterilized and were placed over solid nutrient medium to assess CSC of precolonized substrates by F. r~dum. In a separate experiment the inoculum of F. udum was diluted with that of the test microorganisms, separately, in the same way as used in the Cambridge method. The control was set with pure inoculum of F. udum diluted in the same proportions with sterilized sand +3:/i maize meal. Then the competitive ~prophytic ~o~oni~t~on of virgin pigeon-pea substrate by F. udum was recorded.
RESULTS f@ecr
of’sail
AND DISCUSSION
temperature,
pH and moisture
The most suitable temperature for substrate colonization by F. udum was 22 k 2’C followed by 30 j, 2°C (Table 1). Even at 15 _t 2°C the C,, value was 30, but at 40 + 2°C it was only about 1. Active colonization of the substrate by C. lagopus and A. nidulans was recorded at higher temperatures and thus may have suppressed colonization by F. udum.
UPADHYAY
Upadhyay and Rai (1980) have observed that C. lugopus suppressed the saprophytic colonization of pigeon-pea substrate more effectively at 30°C than at 22°C. In addition to this other microflora including some thermophiiic or thermotolerant microorganisms might grow at high temperature and suppress the activity of F. u&m. A. nidulans has been reported as a thermotolerant fungus in competition with other microflora (Upadhyay and Rai, 1978). High competitive saprophytic colonization of the host substrate by F. udum was noted at pH 5, 7 and 9 (C,, values 99, 100 and 100, respectively). Very low values occurred at pH 4 (C,, value 0, Table 1). At Iow pH A. nidulans, A. terreus and A. ,fluws colonized rather than F. udum. It was recorded that pH of the field soil, infested with F. udum, was slightly alkaline (pH 7.3-7.5) and, therefore, it seems that F. udurn colonizes well in slightly alkaline soil. Soil moisture had relatively little effect over the range 5-307: tested, though CSC by F. udum was best at 15 and 25’:: soil moisture levels. Eflect qf,fungicides The competitive saprophytic colonization of substrate by F. udum decreased as the concentration of the fungicides was increased (Table 1). At various concentrations of different fungicides the substrate units were also colonized by other microorganisms. At I .O’,/, concentration Aspergillus luchuensis Inui, Acremonium sp., Penicillium citrinum and Mortierellcl s~~ti/is~~i~na Oudemans were frequently recorded from the root bits in the case of difolatan, R~i~~~p~~~ nigricans Ehrenberg, Tr~~hap~~ror~ sp. and a white sterile fungus in the case of bavistin and Aspergillus niger, A. jlaous and Cladosporium cludosporioides (Fresen.) de Vries in the case of dithane Z-78. A decrease in the competitive saprophytic colonization of root segments by F. udum due to fungicidal treatment may be attributed to the toxic effect of the fungicides. The other reason may be ascribed that at high concentration a few species mentioned above also colonize the substrate possibly due to their tolerance of fungicides and compete with F. udum. Several other fungi have been reported to possess selective tolerance of fungicides (Gibson, 1953; Greenaway et al., 1974). i@ects oj’ herbicides Both herbicides significantly (P = 0.01) inhibited the saprophytic colonization of substrate by F. udum at 1.0% concentration (C,, value, 0 for each), and machete also had a marked effect at 0. l:‘, concentration (Cse = 30, Table 1). These effects are attributed to toxicity towards F. udum as well as the selective tolerance of fungi to the herbicides. l?ffect
qf urea
Urea at all concentrations promoted CSC by F. udum. This effect might be because better availability of nitrogen supports better growth and activity of the test fungus. Garrett (1967) noted a prolonged survival of Gaeumannomyces gruminis in the presence of inorganic nitrogen or in soil rich in nitrogenous organic matter, which was confirmed later by Chambers and Flentje (1969). However, Garrett (1966) pointed out that whether supplementary nitrogen
CSC of pigeon-pea by F. udum Table
I89
I. Effects of soil physical factors, fungicides colonization
and herbicides on competitive substrate by F. udum (in ?;,)
of pigeon-pea
saprophytic
1noculum:field soil lOO:O 1
2
98:2
90: 10
3
Moisture 5 15 25 30
IO:90
2:98
0: 100
5
6
7
8
9
80 90 92 70
79 76 80 45
32 56 72 25
99 100 100 94
26 75 96 82
8 40 90 74
0 34 70 71
0 99 100 100
26 100 73 2
14 83 62 2
4 71 60 0
30 100 iO0 I
78 52 20 90
64 44 I4 80
60 30 12 72
100 99 0 100
74 32 IO 90
70 28 IO 82
68 20 8 70
100 30 0 100
68 52 I3 95
64 42 I2 82
50 35 IO 68
100 99 0 100
56 25 I4 78
38 I8 8 60
24 15 0 56
36 20 IO 75
25 10 6 62
22 IO 0 60
30 1 0 100
94 92 100 90
82 86 94 87
75 87 90 67
100 100 100 100
4
Eftcr
of’soil
c5”
due
50:50
moisture
(%) 100 100 100 100
PH 4 5 7 9 Temperature 15&2 22 * 2 30 + 2 40 + 2 Concentration 0.1 0.5 1.0 Control
32 95 100 100 (’ C)
85 100 92 98 100 100 100 100 100 89 100 92 Eflect of’.voil pH 24 23 21 90 95 90 100 100 100 90 100 100 IT&cl 0( soil temperature
100 100 100 60
100 100 100 40 IZffkcl
100 90 48 100
100 86 40 100 Eject 100 68 38 100
47 100 100 100 100 100 4 IO of‘ difblaian
(y,) 92 70 34 100 q/ dithanr 96 68 28 100
0.1 0.5 1.0 Control
100 84 42 100
0.1 0.5 1.o Control
100 78 30 100
0.1 0.5 I.0 Control
95 63 37 100
0.1 0.5 1.0 Control
80 65 35 100
100’ 76 25 100 Efltct 90 60 25 100 &$YY 72 38 30 100
0.1 0.5 I .o Control
100 100 100 100
100 100 100 100
Efecl
84 60 26 100 Z- 7X 78 48 14 100
of‘ haoistin
81 90 58 70 I5 24 100 100 of 2,4-D 78 82 45 58 20 25 96 96 q/‘muchrre 40 65 32 38 I6 26 90 100
Efxr
of
100 100 100 100
promotes saprophytic survival or not depends on the Cellulolysis adequacy index (CAI) of the fungus. He found that an index more than unity indicates that a fungus will achieve maximum longivity of survival without supplementary nitrogen. Helminthosporium sativum had a highest index (3.5) amongst the fungi tested by him which was the reason of its shortened survival in presence of supplementary nitrogen. However, the survival of G. gruminis having the CA1 lowest or below unity (0.59) was promoted by supplementary nitrogen. The favourable effect of nitrogen in soil on saprophytic colonization of substrate by a number of fungi has been noted by Butler (1953a,b,
94.5 30.0 0.0 99.0
urea 100 100 100 100
1959) Mater (1961) and Tiwari (1971). We suggest that nitrogenous fertilizers should not be applied to fields infested with F. udum for cultivation of pigeonpea without any crop rotation. The roots of pigeonpea also contribute nitrogen in soil through root nodules which could help in increasing the inoculum of the test pathogen. Competit&e saprophytic root pieces by F. udum Saprophytic
of‘ precolonized
colonization of the substrate by F. to be considerably reduced when it been colonized by some other fungi. P.
udum was found
had already
colonization
190
BHARAT RAI and R. S. UPADHYAY Table 2. Percent
competitive
saprophytic
colonization F. udum
1noculum:field Test microbes Aspergillus Jarus A. niger A. terreus Coprinus tagopus Paputaspora sp. Penicillium citrinum Rkizoctonia solani Trichoderma ciride Micromonospora glohosa Yellow bacterium Control
of precolonized
pigeon-pea
substrate
soil
,-
L
1oo:o
98:2
90: 10
so:50
10:90
2:98
0: 100
val:0e
80 64 15 100 100
61 35 52 98 100
41 20 42 78 95
25 6 30 55 80
18 0 15 38 75
0 0 4 14 28
0 0 0 0 0
6 1 6 70 94
50
34
23
8
0
0
0
0
100
90
16
55
38
17
0
70
90
70
52
34
18
8
0
30
60 65 100
40 46 100
31 30 100
17 26 100
0 16 90
0 0 82
0 0 70
1 1 100
citrium had the greatest effect (CsOvalue = 0) followed by a significant reduction in the case of A. niger, M. globosa, A. .ftacus, A. terreus and T. viride (C,, value in each case less than 7 except for T. uiride where the C,, value was 30; Table 2). The reduction of the saprophytic colonization of the substrate by F. udum might have also occurred due to its competition with other individual fungi that had colonized the substrate. The possibility of F. udum being present inside the substrate units but unable to reveal itself due to other fungi preventing it from emerging from the substrate onto agar may not be ruled out and requires further investigation. In the case of root segments that had been colonized by Papulaspora sp., C. lagopus and R. solani the CSC by F. udum was reduced only slightly (C,, value in each case more than 85; Table 2). The decrease in ability of F. udum to colonize the pigeon-pea substrates already colonized by the aforesaid microflora may be attributed to their antagonistic behaviour towards F. udum. The micro-
organisms that suppressed CSC by F. udum were antagonistic in in vitro studies also (Upadhyay, 1979). Their toxic metabolites or nutrient depletion by them during colonization of substrate may be responsible for inhibiting CSC by F. udum. In contrast, fungi like Papulaspora sp., C. lagopus and R. solani showed insignificant suppression of CSC, and these fungi have been found to be less effective against F. udum in a separate study on antagonism in cultural conditions (Upadhyay, 1979). Microbial competition for nutrients or space, and the presence of toxic substances play a key role in antagonism. These factors are chiefly governed by the physico-chemical nature of the environment (Garrett, 1963; Burgess and Griffin, 1967). Competitive saprophytic colonization by F. udum inoculum mixtures with other microorganisms
(at 25 + 1°C) in different test microorganisms
F. udum :Test microbes
Aspergillus Jtacus A. niger A. terreus Coprinus lagopus Papulaspora sp. Pencillium citrinum Rhizoctonia solani Trichoderma oiride Micromonospora globosa Yellow bacterium Control
in
When the inoculum of F. udum was diluted with inoculum of the test microbes separately, CSC was suppressed to different degrees. M. globosa caused a
Table 3. Percent colonization of pigeon-pea substrate by F. udum inoculum ratios of F. udum diluted with the inocula of different Test microbes
by
1oo:o
98:2
9O:lO
50:50
100 100 100
54 68 76
30 36 32
22 20 18
6 6 8
100 100
95 82
81 74
45 50
100
60
42
100
94
100 100 100 100
10:90
C 2:98
0: 100
vaZe
4 0 6
0 0 0
6 6 6
18 26
12 22
0 0
30 50
16
10
6
0
6
70
52
30
22
0
70
90
68
38
26
18
0
30
40 92 100
32 16 100
18 62 95
8 44 80
4 30 72
0 0 0
1 70 99
191
CSC of pigeon-pea by F. udum substantial
in CSC (C,, = 1, Table 3). A. terreus and P. citrinum also reduced the CSC (C,, value for each = 6). These fungi have been observed to suppress the growth of F. udum (Upadhyay, 1979). The degree of reduction of CSC was less in the case of C. lagopus, Papulaspora sp., R. solani, T. viride and a yellow bacterium (C,, values, 30, 50, 70, 30 and 70, respectively; Table 3). These microbes were less antagonistic against F. udum in cultural studies (Upadhyay, 1979). The results in general show that F. udum is a vigorous saprophytic colonizer of pigeon-pea substrate in competition with other soil microflora. The colonization of root fragments decreased insignificantly by increasing the dilution of pure inoculum of F. udum; 50-70’y0 of substrate units were colonized by it even in the field soil unamended with F. udum which indicates that the pathogen has a high competitive saprophytic ability. During our investigation an attempt was made to evaluate the factors which suppress the competitive saprophytic colonization of pigeon-pea (host) substrate by the wilt pathogen. Garrett (1963) distinguished saprophytic survival from saprophytic colonization. Saprophytic survival permits the survival of parasites in dead host tissue that they have invaded and occupied when the host tissues were alive whereas saprophytic colonization is the invasion of dead host tissues in competition with saprophytic fungi. The former is significant for the wilted plants of pigeon-pea and the latter for uninfested plant substrates. When the substrate units are colonized first by F. udum it becomes difficult for other microflora to replace it (unpublished data). However, as is evident from Tables 2 and 3, saprophytic colonization is remarkably reduced by an antogonistic microflora. Thus field sanitation by removing wilted pigeonpea plants and manipulation of the CSC of F. udum by introducing an antagonistic microflora into soil or by any other means may be useful to reduce the population of F. udum in soil. Soil conditions may also be altered to reduce the saprophytic activity of the root parasite. The soil temperature and moisture can be controlled to some extent by varying the date of sowing and soil reaction by use of lime or sulphur (Garrett, 1956). Crop rotation has also been suggested as a means to reduce the inoculum of F. udum (Upadhyay and Rai, 1982).
niger,
reduction
A. jaws,
A.
Acknowledgements-We thank the Head of the Botany Department, Banaras Hindu University for laboratory facilities. RSU is grateful to the Council of Scientific and Industrial Research, New Delhi for providing him with a Research Associateship. REFERENCES
Burgess W. and Griffin D. M. (1967) Competitive saprophytic colonization of wheat straw. Annals of Applied Biology 60, 137-142.
Butler F. C. (1953a) Saprophytic behaviour of some cereal root-rot fungi. I. Saprophytic colonization of wheat straw. Annals of Applied Biology 40, 284297. Butler F. C. (1953b) Saprophytic behaviour of some cereal root-rot fungi. II. Factors influencing the saprophytic colonization of wheat straws. Annals of Applied Biology 40, 298-304.
Butler F. C. (1959) Saprophytic behaviour of some cereal root-rot fungi. IV. Saprophytic survival in soils of high and low fertility. Annals of Applied Biology 47, 28-36. Chambers S. C. and Flentje N. T. (1969) Relative effects of soil nitrogen and soil organisms on survival of Ophiobolus graminis. Australian Journal of Biological Sciences 22, 275-278.
Garrett S. D. (1950) Ecology of the root inhabiting fungi. Biological Reviews 25, 220-254.
Garrett S. D. (1956) Biology of Root-Infecting Fungi. Cambridge University Press. Garrett S. D. (1963)Soil Fungi and Soil Fertility. Pergamon Press, Oxford. Garrett S. D. (1965) Toward biological control of soil-borne plant pathogens. In Ecology of Soil-borne Plant Pathogens (K. F. Baker and W. C. Snyder, Eds), pp. 4-17. Murray, London. Garrett S. D. (1966) Cellulose-decomposing ability of some cereal foot-rot fungi in relation to their saprophytic survival. Transactions of the British Mycological Society 49, 57-68.
Garrett S. D. (1967) Effect of nitogen level on survival of Ophiobolus graminis in pure culture on cellulose. Transactions of the British Mycological Society 50, 519-524.
Garrett S. D. (1975) Cellulolysis rate and competitive saprophytic colonization of wheat straw by foot-rot fungi. Soil Biology & Biochemistry 7, 323-327. Gibson I. A. S. (1953) Crown rot, a seedling disease of groundnuts caused by Aspergilius niger. II. An anomalous effect of organomercurial seed dressings. Transactions of the British Mycological Society 36, 324-334.
Greenaway W., Cripps A. and Ward S. (1974) Resistance to organo-mercury by penicillia isolated from cereal seed. Transactions 137-141.
of the British
Mycological
Society
63,
Mater R. C. F. (1961) Saprophytic colonization of wheat straw by Cercosporella herpotrichoides Fron. and other fungi. Annals of Applied Biology 49, 152-164. Park D. (1965) Survival of microorganisms in soil. In Ecology of Soil-borne Plant Pathogens (K. F. Baker and W. C. Snyder, Eds), pp. 82-97. Murray, London. Tiwari M. N. (1971) Studies on Heiminthosporium tetramera Mck. of rice plant with reference to its competitive saprophytic ability. Ph.D. Thesis, Banaras Hindu University, India. Upadhyay R. S. (1979)Ecological studies on Fusarium udum Butler causing wilt disease of pigeon-pea. Ph.D. Thesis, Banaras Hindu University, India. Upadhyay R. S. and Rai B. (1978) Tolerance to higher temperatures by Aspergillus nidulans in competition with other soil fungi. Proceedings of the National Academy of Sciences, India, p. 77 (abstract). Upadhyay R. S. and Rai B. (1980) Interrelationship between Coprinus lagopus and Fusarium udum in soil in relation to competitive saprophytic ability and microbial antagonism. Plant and Soil 56, 169-173. Upadhyay R. S. and Rai B. (1982) Ecology of Pusarium udum causing wilt disease of pigeon pea: Population dynamics in the root region Transactions of the British Mycological Society 78, 209-220.
0038-0717/83/020187-05$03.00/O Copyright C 1983 Pergamon Press Ltd
COMPETITIVE SAPROPHYTTC COLONIZATION OF PIGEON-PEA SUBSTRATE BY PUSARZUM UDUA4 IN RELATION TO ENVIRONMENTAL FACTORS, CHEMICAL TREATMENTS AND MICROBIAL ANTAGONISM BHARAT RAI and R. S. UPADHYAY Department
of Botany,
Banaras
Hindu
University,
Varanasi
221005,
India
(Accepted 20 July 1982)
Summary-The competitive saprophytic colonization (CSC) of pigeon-pea substrates by Fusurium udum was studied in relation to environmental factors, chemical treatments and microbial antagonism. CSC was best at 22”C, good at 3O”C, but at 40°C the substrate was vigorously colonized by Coprinus lagopus and Aspergillus niduluns which suppressed colonization by F. udum. High competitive saprophytic ability of F. udum was noted at pH 7 and 9 at soil moisture contents between 5 and 30%. At pH 4 and 5 F. udum was replaced by A.javus and A. nidulans. The CSC of F. udum decreased in the soil-sand inoculum mixture amended with the fungicides bavistin, dithane Z-78 and difolatan. The herbicides 2,4-D and machete also reduced CSC by F. udum whereas urea promoted it. Colonization of pigeon-pea substrate by F. udum was highly suppressed by antagonism from Penicillium citrinum, A. niger, Micromonospora globosa, A.Jlaous, A. terreus and Trichoderma Gride when these were used in inoculum mixtures with F. udum or when substrates had already been colonized by them. Otherwise F. udum was shown to have a high competitive saprophytic ability.
INTRODUCTION Fusarium
udum Butler is a soil-inhabiting pathogenic root-infecting fungus which causes wilt disease of pigeon-pea (Cujanus cujan (L.) Millsp.). The extent of ability of a soil-borne plant pathogen to survive saprophytically in the absence of its host depends on its competitive saprophytic ability; this may be assessed by competitive saprophytic colonization of substrate units buried in graded series of inocu1um:soil dilutions (Garrett, 1950). Competitive saprophytic ability in relation to biological control of soil-borne diseases has been discussed by Garrett (1965) and Park (1965). We have investigated the competitive saprophytic colonization of pigeon-pea substrate by F. udum in relation to moisture, pH, temperature, chemical treatments and microbial anatgonism.
MATERIALS AND METHODS
The Cambridge method (Garrett, 1963) was followed. Pure inoculum of the test pathogen was prepared in 500 ml conical flasks each containing 200 g sterilized acid-washed sand + 3% maize meal. Each flask was inoculated with three blocks (10 mm dia) of agar culture of F. udum. Moisure in the sand was maintained at 25’j/, and its pH was adjusted to 6.5. The flasks were incubated at 25 k 2°C for 10 days. The flasks were shaken periodically so as to maintain a homogeneous continuum of propagules. The inoculum-sand mixture of F. udum, prepared in this way, was mixed with field soil in different proportions (w/w), i.e. lOO:O, 98:2, 90:10, 50:50, 10:90, 2:98 187
and 0: 100; the first and last serving as respective controls for inoculum and field soil. The soil sample was collected from a pigeon-pea field situated near Broacha Hostel, Banaras Hindu University Campus. The soil contained a high resident population of F. udum due to wilt disease of pigeon-pea in the field (0.61-1.40 x lo3 g-’ dry soil; Upadhyay and Rai, 1982) and this was confirmed by repeated isolation of its colonies on Martin’s agar medium (Upadhyay, 1979). Moisture in the inoculum-soil mixture was maintained at 25 + 3% except where mentioned otherwise. The substrate used included roots and branches of stem from healthy pigeon-pea plants collected from the field at harvest. Fifty pieces (2cm length and 0.2-0.5 cm dia) of sterilized substrate were buried in 250 ml conical flasks containing 1oog of inoculum-soil mixture. The flasks were held at 25 f 1°C for 4 weeks and then the substrate units were taken out and washed 10-12 times in sterile distilled water. The washed pieces were surface sterilized with 0.1% HgC12 (in 10% alcohol) for 1 min then rinsed in sterile distilled water. The pieces were then blotted between the folds of sterile blotting papers and were transferred to Petri dishes (5 pieces/dish) containing 20 ml Czapek-Dox agar medium. Each treatment was replicated three times. All Petri dishes were held at 22 & 2°C for 6 days and the percentage colonization of root/shoot substrates by the test fungus was recorded. From graphs of per cent colonization against inoculum dilution the api.e. the perproximate C,, value was calculated, centage of non-sterile soil permitting colonization of 50% of the substrate units by F. udum (Garrett, 1975).
BHAKAT RAI
188
Non-biological
and R. S.
,fbctors studied
The effects of the following were studied in triplicate sets of all soil--inocuIum mixtures: ---Soil moisture at 5, 15, 25 and 30% moisture {oven-dry basis) maintained with sterile distilled water. -Soil reaction adjusted to pH 4, 5, 7 and 9 by dilute acid or alkali. --Soil temperature by incubation at 15 & 2,22 + 2, 30 2 2 and 40 k 2°C. -The three fungicides dithane Z-78 (75% zinc ethylene ~j.~-dithio~arbamate and 25% inert ingredients, Indofil Chemicals Ltd, Bombay), difolatan (80% cis,N-( I ,1,2,2-tetrachoroethyl) (thio) 4-cyclohexane I, dicarboxymide, Rallies India Ltd, Bombay) and carbamoylbenzimidazole, bavistin (7%~ Methoxy BASF India Ltd. Bombay). 0.1,O.S and 1.O?/, concentrations of the fungicides were at 20% moisture level. Herbicides. 0.1, 0.5 and 1.0% concentration of 2,4-D (2,4-dichlorophenoxy acetic acid, 80% active ingredient) and machete (2-chloro-2’,6’-diethyl-N(butoxymethyl) acetanilide, 50% active ingradient). Urcu. 0.1. 0.5 and I .O:/, concentrations.
Aspergiks fiarus Link ex Fries, A. niger van Tieghem, A. terreus Thorn, Coprinus lugopus Fr., Pupulasporu sp., Penicillium citrinum Thorn, Rhizoctoniu solani Kiihn, Trichoderma viride Pers ex Gray, Micromonospara glohosa Krass. and one yellow bacterium were used. Pure inocula of these test lni~rooganisms were prepared separately in sterilized sand + 3”i;, maize meal in conical flasks. Four hundred sterile root segments (2cm length and 0.2-0.5cm dia) of pigeon-pea were buried in each flask and were held at 25 & 1°C for 20 days. The root segments were then removed, washed thoroughly and transferred to 250 ml conical flasks containing 100 g different dilutions of sand-inoculum (100, 9X,90, 50, 10 and 2’;d diluted with field soil) of F. udum. The flasks were then held at 25 + 1°C for 4 weeks. Thereafter, the root segments were washed several times with distilled water, surface sterilized and were placed over solid nutrient medium to assess CSC of precolonized substrates by F. r~dum. In a separate experiment the inoculum of F. udum was diluted with that of the test microorganisms, separately, in the same way as used in the Cambridge method. The control was set with pure inoculum of F. udum diluted in the same proportions with sterilized sand +3:/i maize meal. Then the competitive ~prophytic ~o~oni~t~on of virgin pigeon-pea substrate by F. udum was recorded.
RESULTS f@ecr
of’sail
AND DISCUSSION
temperature,
pH and moisture
The most suitable temperature for substrate colonization by F. udum was 22 k 2’C followed by 30 j, 2°C (Table 1). Even at 15 _t 2°C the C,, value was 30, but at 40 + 2°C it was only about 1. Active colonization of the substrate by C. lagopus and A. nidulans was recorded at higher temperatures and thus may have suppressed colonization by F. udum.
UPADHYAY
Upadhyay and Rai (1980) have observed that C. lugopus suppressed the saprophytic colonization of pigeon-pea substrate more effectively at 30°C than at 22°C. In addition to this other microflora including some thermophiiic or thermotolerant microorganisms might grow at high temperature and suppress the activity of F. u&m. A. nidulans has been reported as a thermotolerant fungus in competition with other microflora (Upadhyay and Rai, 1978). High competitive saprophytic colonization of the host substrate by F. udum was noted at pH 5, 7 and 9 (C,, values 99, 100 and 100, respectively). Very low values occurred at pH 4 (C,, value 0, Table 1). At Iow pH A. nidulans, A. terreus and A. ,fluws colonized rather than F. udum. It was recorded that pH of the field soil, infested with F. udum, was slightly alkaline (pH 7.3-7.5) and, therefore, it seems that F. udurn colonizes well in slightly alkaline soil. Soil moisture had relatively little effect over the range 5-307: tested, though CSC by F. udum was best at 15 and 25’:: soil moisture levels. Eflect qf,fungicides The competitive saprophytic colonization of substrate by F. udum decreased as the concentration of the fungicides was increased (Table 1). At various concentrations of different fungicides the substrate units were also colonized by other microorganisms. At I .O’,/, concentration Aspergillus luchuensis Inui, Acremonium sp., Penicillium citrinum and Mortierellcl s~~ti/is~~i~na Oudemans were frequently recorded from the root bits in the case of difolatan, R~i~~~p~~~ nigricans Ehrenberg, Tr~~hap~~ror~ sp. and a white sterile fungus in the case of bavistin and Aspergillus niger, A. jlaous and Cladosporium cludosporioides (Fresen.) de Vries in the case of dithane Z-78. A decrease in the competitive saprophytic colonization of root segments by F. udum due to fungicidal treatment may be attributed to the toxic effect of the fungicides. The other reason may be ascribed that at high concentration a few species mentioned above also colonize the substrate possibly due to their tolerance of fungicides and compete with F. udum. Several other fungi have been reported to possess selective tolerance of fungicides (Gibson, 1953; Greenaway et al., 1974). i@ects oj’ herbicides Both herbicides significantly (P = 0.01) inhibited the saprophytic colonization of substrate by F. udum at 1.0% concentration (C,, value, 0 for each), and machete also had a marked effect at 0. l:‘, concentration (Cse = 30, Table 1). These effects are attributed to toxicity towards F. udum as well as the selective tolerance of fungi to the herbicides. l?ffect
qf urea
Urea at all concentrations promoted CSC by F. udum. This effect might be because better availability of nitrogen supports better growth and activity of the test fungus. Garrett (1967) noted a prolonged survival of Gaeumannomyces gruminis in the presence of inorganic nitrogen or in soil rich in nitrogenous organic matter, which was confirmed later by Chambers and Flentje (1969). However, Garrett (1966) pointed out that whether supplementary nitrogen
CSC of pigeon-pea by F. udum Table
I89
I. Effects of soil physical factors, fungicides colonization
and herbicides on competitive substrate by F. udum (in ?;,)
of pigeon-pea
saprophytic
1noculum:field soil lOO:O 1
2
98:2
90: 10
3
Moisture 5 15 25 30
IO:90
2:98
0: 100
5
6
7
8
9
80 90 92 70
79 76 80 45
32 56 72 25
99 100 100 94
26 75 96 82
8 40 90 74
0 34 70 71
0 99 100 100
26 100 73 2
14 83 62 2
4 71 60 0
30 100 iO0 I
78 52 20 90
64 44 I4 80
60 30 12 72
100 99 0 100
74 32 IO 90
70 28 IO 82
68 20 8 70
100 30 0 100
68 52 I3 95
64 42 I2 82
50 35 IO 68
100 99 0 100
56 25 I4 78
38 I8 8 60
24 15 0 56
36 20 IO 75
25 10 6 62
22 IO 0 60
30 1 0 100
94 92 100 90
82 86 94 87
75 87 90 67
100 100 100 100
4
Eftcr
of’soil
c5”
due
50:50
moisture
(%) 100 100 100 100
PH 4 5 7 9 Temperature 15&2 22 * 2 30 + 2 40 + 2 Concentration 0.1 0.5 1.0 Control
32 95 100 100 (’ C)
85 100 92 98 100 100 100 100 100 89 100 92 Eflect of’.voil pH 24 23 21 90 95 90 100 100 100 90 100 100 IT&cl 0( soil temperature
100 100 100 60
100 100 100 40 IZffkcl
100 90 48 100
100 86 40 100 Eject 100 68 38 100
47 100 100 100 100 100 4 IO of‘ difblaian
(y,) 92 70 34 100 q/ dithanr 96 68 28 100
0.1 0.5 1.0 Control
100 84 42 100
0.1 0.5 1.o Control
100 78 30 100
0.1 0.5 I.0 Control
95 63 37 100
0.1 0.5 1.0 Control
80 65 35 100
100’ 76 25 100 Efltct 90 60 25 100 &$YY 72 38 30 100
0.1 0.5 I .o Control
100 100 100 100
100 100 100 100
Efecl
84 60 26 100 Z- 7X 78 48 14 100
of‘ haoistin
81 90 58 70 I5 24 100 100 of 2,4-D 78 82 45 58 20 25 96 96 q/‘muchrre 40 65 32 38 I6 26 90 100
Efxr
of
100 100 100 100
promotes saprophytic survival or not depends on the Cellulolysis adequacy index (CAI) of the fungus. He found that an index more than unity indicates that a fungus will achieve maximum longivity of survival without supplementary nitrogen. Helminthosporium sativum had a highest index (3.5) amongst the fungi tested by him which was the reason of its shortened survival in presence of supplementary nitrogen. However, the survival of G. gruminis having the CA1 lowest or below unity (0.59) was promoted by supplementary nitrogen. The favourable effect of nitrogen in soil on saprophytic colonization of substrate by a number of fungi has been noted by Butler (1953a,b,
94.5 30.0 0.0 99.0
urea 100 100 100 100
1959) Mater (1961) and Tiwari (1971). We suggest that nitrogenous fertilizers should not be applied to fields infested with F. udum for cultivation of pigeonpea without any crop rotation. The roots of pigeonpea also contribute nitrogen in soil through root nodules which could help in increasing the inoculum of the test pathogen. Competit&e saprophytic root pieces by F. udum Saprophytic
of‘ precolonized
colonization of the substrate by F. to be considerably reduced when it been colonized by some other fungi. P.
udum was found
had already
colonization
190
BHARAT RAI and R. S. UPADHYAY Table 2. Percent
competitive
saprophytic
colonization F. udum
1noculum:field Test microbes Aspergillus Jarus A. niger A. terreus Coprinus tagopus Paputaspora sp. Penicillium citrinum Rkizoctonia solani Trichoderma ciride Micromonospora glohosa Yellow bacterium Control
of precolonized
pigeon-pea
substrate
soil
,-
L
1oo:o
98:2
90: 10
so:50
10:90
2:98
0: 100
val:0e
80 64 15 100 100
61 35 52 98 100
41 20 42 78 95
25 6 30 55 80
18 0 15 38 75
0 0 4 14 28
0 0 0 0 0
6 1 6 70 94
50
34
23
8
0
0
0
0
100
90
16
55
38
17
0
70
90
70
52
34
18
8
0
30
60 65 100
40 46 100
31 30 100
17 26 100
0 16 90
0 0 82
0 0 70
1 1 100
citrium had the greatest effect (CsOvalue = 0) followed by a significant reduction in the case of A. niger, M. globosa, A. .ftacus, A. terreus and T. viride (C,, value in each case less than 7 except for T. uiride where the C,, value was 30; Table 2). The reduction of the saprophytic colonization of the substrate by F. udum might have also occurred due to its competition with other individual fungi that had colonized the substrate. The possibility of F. udum being present inside the substrate units but unable to reveal itself due to other fungi preventing it from emerging from the substrate onto agar may not be ruled out and requires further investigation. In the case of root segments that had been colonized by Papulaspora sp., C. lagopus and R. solani the CSC by F. udum was reduced only slightly (C,, value in each case more than 85; Table 2). The decrease in ability of F. udum to colonize the pigeon-pea substrates already colonized by the aforesaid microflora may be attributed to their antagonistic behaviour towards F. udum. The micro-
organisms that suppressed CSC by F. udum were antagonistic in in vitro studies also (Upadhyay, 1979). Their toxic metabolites or nutrient depletion by them during colonization of substrate may be responsible for inhibiting CSC by F. udum. In contrast, fungi like Papulaspora sp., C. lagopus and R. solani showed insignificant suppression of CSC, and these fungi have been found to be less effective against F. udum in a separate study on antagonism in cultural conditions (Upadhyay, 1979). Microbial competition for nutrients or space, and the presence of toxic substances play a key role in antagonism. These factors are chiefly governed by the physico-chemical nature of the environment (Garrett, 1963; Burgess and Griffin, 1967). Competitive saprophytic colonization by F. udum inoculum mixtures with other microorganisms
(at 25 + 1°C) in different test microorganisms
F. udum :Test microbes
Aspergillus Jtacus A. niger A. terreus Coprinus lagopus Papulaspora sp. Pencillium citrinum Rhizoctonia solani Trichoderma oiride Micromonospora globosa Yellow bacterium Control
in
When the inoculum of F. udum was diluted with inoculum of the test microbes separately, CSC was suppressed to different degrees. M. globosa caused a
Table 3. Percent colonization of pigeon-pea substrate by F. udum inoculum ratios of F. udum diluted with the inocula of different Test microbes
by
1oo:o
98:2
9O:lO
50:50
100 100 100
54 68 76
30 36 32
22 20 18
6 6 8
100 100
95 82
81 74
45 50
100
60
42
100
94
100 100 100 100
10:90
C 2:98
0: 100
vaZe
4 0 6
0 0 0
6 6 6
18 26
12 22
0 0
30 50
16
10
6
0
6
70
52
30
22
0
70
90
68
38
26
18
0
30
40 92 100
32 16 100
18 62 95
8 44 80
4 30 72
0 0 0
1 70 99
191
CSC of pigeon-pea by F. udum substantial
in CSC (C,, = 1, Table 3). A. terreus and P. citrinum also reduced the CSC (C,, value for each = 6). These fungi have been observed to suppress the growth of F. udum (Upadhyay, 1979). The degree of reduction of CSC was less in the case of C. lagopus, Papulaspora sp., R. solani, T. viride and a yellow bacterium (C,, values, 30, 50, 70, 30 and 70, respectively; Table 3). These microbes were less antagonistic against F. udum in cultural studies (Upadhyay, 1979). The results in general show that F. udum is a vigorous saprophytic colonizer of pigeon-pea substrate in competition with other soil microflora. The colonization of root fragments decreased insignificantly by increasing the dilution of pure inoculum of F. udum; 50-70’y0 of substrate units were colonized by it even in the field soil unamended with F. udum which indicates that the pathogen has a high competitive saprophytic ability. During our investigation an attempt was made to evaluate the factors which suppress the competitive saprophytic colonization of pigeon-pea (host) substrate by the wilt pathogen. Garrett (1963) distinguished saprophytic survival from saprophytic colonization. Saprophytic survival permits the survival of parasites in dead host tissue that they have invaded and occupied when the host tissues were alive whereas saprophytic colonization is the invasion of dead host tissues in competition with saprophytic fungi. The former is significant for the wilted plants of pigeon-pea and the latter for uninfested plant substrates. When the substrate units are colonized first by F. udum it becomes difficult for other microflora to replace it (unpublished data). However, as is evident from Tables 2 and 3, saprophytic colonization is remarkably reduced by an antogonistic microflora. Thus field sanitation by removing wilted pigeonpea plants and manipulation of the CSC of F. udum by introducing an antagonistic microflora into soil or by any other means may be useful to reduce the population of F. udum in soil. Soil conditions may also be altered to reduce the saprophytic activity of the root parasite. The soil temperature and moisture can be controlled to some extent by varying the date of sowing and soil reaction by use of lime or sulphur (Garrett, 1956). Crop rotation has also been suggested as a means to reduce the inoculum of F. udum (Upadhyay and Rai, 1982).
niger,
reduction
A. jaws,
A.
Acknowledgements-We thank the Head of the Botany Department, Banaras Hindu University for laboratory facilities. RSU is grateful to the Council of Scientific and Industrial Research, New Delhi for providing him with a Research Associateship. REFERENCES
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Mater R. C. F. (1961) Saprophytic colonization of wheat straw by Cercosporella herpotrichoides Fron. and other fungi. Annals of Applied Biology 49, 152-164. Park D. (1965) Survival of microorganisms in soil. In Ecology of Soil-borne Plant Pathogens (K. F. Baker and W. C. Snyder, Eds), pp. 82-97. Murray, London. Tiwari M. N. (1971) Studies on Heiminthosporium tetramera Mck. of rice plant with reference to its competitive saprophytic ability. Ph.D. Thesis, Banaras Hindu University, India. Upadhyay R. S. (1979)Ecological studies on Fusarium udum Butler causing wilt disease of pigeon-pea. Ph.D. Thesis, Banaras Hindu University, India. Upadhyay R. S. and Rai B. (1978) Tolerance to higher temperatures by Aspergillus nidulans in competition with other soil fungi. Proceedings of the National Academy of Sciences, India, p. 77 (abstract). Upadhyay R. S. and Rai B. (1980) Interrelationship between Coprinus lagopus and Fusarium udum in soil in relation to competitive saprophytic ability and microbial antagonism. Plant and Soil 56, 169-173. Upadhyay R. S. and Rai B. (1982) Ecology of Pusarium udum causing wilt disease of pigeon pea: Population dynamics in the root region Transactions of the British Mycological Society 78, 209-220.