Soil Bioi. Biochem. Vol. 12. pp. 531 to 536 Q Pergamon Press Ltd 1980. Printed in Great
ao38-0717/80/l
lol~o53lsoz.~~
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THE RESPONSE OF CYLINDROCLADIUM SOIL FUNGISTASIS
CONIDIA TO
DON A. ROTH*and GARY J. GRIFFIN Department of Plant Pathology and Physiology, Virginia Polytechnic Inst. and State Univ., Blacksburg, VA 24061, U.S.A. (Accepted 1 June 1980) observation of washed conidia of C~~~ndroc~ad~um scopQriu~ on non-sterile soils, air dried and rewetted immediately before deposition of eonidia. indicated that peak ge~ination (33-58%) occurred after 24 h incubation at 26°C. Peak germination on continually moist soils was lower (l&260/,) than on rewetted soils. Lysis of germ tubes and germinating conidia on continually moist soils at 26°C was evident with 48 h. Conidia did not germinate on continually moist soils at 6°C and lysis did not become apparent until 168 h. Conidia germinated at a high level (93-99x) in axenic culture in the absence of exogenous C and N sources. The inhibition of conidial germination on soils may be attributed, in part, to the presence of soil volatiles. Germination of conidia placed on washed agar disks and exposed to volatiles from four soils ranged from 51 to 86% of the no-soil controls. Addition of carbon (13 ng C per conidium as glucose) and nitrogen (65 pg N ng- ‘C as NH&l) nullified the inhibitory effect of the soil volatiles. Germinability assayed on a selective medium at 26°C of conidia in artificially infested soils (approximately 10’ conidia g- ’ soil) decreased progressively during incubation at 26°C from 1 week to 4 months. No germinable conidia were recovered from artificially infested soils after 2 months incubation at 6°C. Conidia of C. jforidanum and C. crotulariae responded similarly to C. sc~puriumin many assays.
Summary-Direct
INTRODUmON
stasis (R. H. Morrison, unpublished M.S. thesis, University of Minnesota). We present evidence that conidia of C. crotalariae, C. Jloridanum Sobers and Seymour and C. scoparium Morgan are sensitive to soil fungistasis and examine possible mechanisms explaining the observed inhibition of conidial germination. Information on conidium survival in soil is also provided. A preliminary report has been presented (Roth and Griffin, 1978).
Several hypotheses explaining the mechanisms responsible for the widespread inability of viable fungal propagules to germinate in non-sterile, non-amended soil (soil fungistasis) have been presented but none has met with general acceptance (Lockwood, 1977; Griffin and Roth, 1979). Fungal propagules which are insensitive to soil fungistasis may aid in elucidating the importance of the proposed mechanisms. Recently, Hwang and Ko (1974, 1976) indicated that conidia of C~l~necfria crotulariae (Loos) Bell & Sobers [perfect stage of ~ylindrocl~i~ crotalariue
(Loos) Bell & Sobers] germinated S@-89% on the surface of non-sterile. non-amended soils and 60% when conidia were mixed into the soil at high spore density (a condition that usually suppresses spore germination). Thus, they concluded that C. crotuluriae conidia are insensitive to soil fungistasis. Similarly, D. T. Krigsvold (unpublished data) found that microsclerotia of C. crotalariae, grown in a glucose-casein-hydrolysate medium, were insensitive to soil fungistasis in three soils. Previously, the only fungal spores shown to be insensitive to soil fungistasis (> 90”/, germination on soil) were ascospores of Neurospora tetrusperma and uredospores of Puccinia eoronuru (Ko and Lockwood, 1967). However, the use of non-soil fungi in fungistasis assays has been criticized and may result in erroneous conclusions (Watson and Ford, 1972; Griffin and Roth, 1979). Thus, the demonstration that spores of the soil-borne fungus C. crotahiae, are relatively insensitive to soil fungistasis is potentially of importance. Previous work indicated that C. jloridanum conidia are sensitive to soil fungi* Present address: Plant Science Division, University of Wyoming, Laramie. WY 82071. U.S.A.
MATERIALS AND METHODS
Preparation of conidia Cylindrocladium scoparium and C. Jtoridanum were isolated from naturally-infested black walnut seedbed soils, single-spored and maintained by multipoint transfers on Czapek-Dox (Difco) agar. Cylindrocladium crotulariae was isolated from a naturally-infested peanut field soil, single-spored and maintained by multipoint transfers on acidified potato-dextrose agar, PDA (Difco). All cultures were kept at 26-28°C in room light. Conidia, 6-8 days old, were harvested aseptically from cultures with a sterile, inorganic salt solution (B solution) composed of 1Om~ sodium phosphate, O‘OS%KCl and 0.05% MgSO,*?H,O (pH 5.7). Conidial suspensions were filtered through double-layered cheesecloth (grade 60) and washed three to four times with 1.50-ml portions of B solution by filtration in the top portion of a Millipore filter apparatus (pore size 0.45 m), to remove exogenous C from the growth medium. Conidia were counted in
a haemacytometer and suspensions were adjusted with sterile B solution to 5 x lo4 conidia ml-‘, unless otherwise indicated.
531
532
DON A. ROTH and GARY J. GRIFFIU
Soils
The modified&oil-emanationagar (SAE) method (Griffin et a/.. 1975) was used to determine the influence of soil volatiles on spore germination. Inoculum consisted of 20 ~1 of a 3 x lo4 ml- ’ conidial suspension in B solution or B solution plus glucose and NH,Cl per washed agar disk. Percentage conidium germination on the washed agar disks was assayed after 16 h incubation. The sterileeNucleopore_agar-diffusion (SNAD) method (Hora et (II., 1977) was used to assay for nutrients and inhibitors in soil available to influence spore germination. Inoculum in this experiment consisted of 20 ~1 of a 4 x I Oh conidia ml-’ suspension per washed agar disk. At this density, no germination of Cylindrocladium conidia was found in the absence of exogenous C substrates. Washed agar disks placed on Nucleopore filters, floating on sterile doubly glassdistilled water, in Petri plates served as controls. After 8 h incubation, germination was stopped by pipetting a drop of 10% Roccal (50% alkyl dimethylbenzylammonium chlorides) onto each disk. Percentage germination in the SEA and SNAD experiments was based on counts of 300 conidia (100 per disk) in each of two replications. Germination on non-washed agar disks in the SNAD experiments was assayed also. Survival of washed Cylindroc/adium conidia in artificially-infested soils incubated at 6 and 26°C was studied. Washed conidia (approximately 1 x lo4 conidia g- ’ soil. oven-dry basis) were thoroughly mixed for 30 min into moist soils (pH 5.5 and 5.8), incubated in beakers, covered with aluminium foil, in covered water baths. Conidium populations were assayed by a standard soil-dilution method (log soil sample used to make a 10m3 dilution). One ml of the soil dilution was pipetted onto each of 10 plates of sucrose-thiabendazole medium, selective for Cylindrocladium spp (Krigsvold and Griffin, 1975). Population counts were based on numbers of Cvlindrocladim colonies developing on 10 plates per s&l sample.
Soils used were a clay loam (pH 6.1). sandy loam (pH 5.8) and two loamy sands (pH 5.1 and 5.5). Soils of pH 5.8 and pH 5.5 were collected from black walnut forest-nursery seedbeds. Soils were stored airdried at 26”C, and uniformly rewetted with distilled water to approximately SCrlOO’~ of the -0.33 bars (field capacity) percentage values at least 5 days before beginning each experiment, unless otherwise indicated. Assay procedures
Germination of washed conidia on non-sterile, nonamended soils was assayed by a modification of the Lingappa and Lockwood (1963) method. Approximately 20g of soil was placed in small Petri dishes (15 x 50 mm), the surface smoothed with a spatula, and 0.2 ml conidial suspension pipetted onto the surface. Following incubation for 12, 24,48, 72 and 168 h at 6 and 26”C, the soil was stained with acid fuchsin in lactophenol and smeared on a glass slide for observation. Germination of washed conidia in non-sterile soil was assayed also by the buried-membrane-filter method (Adams, 1967). Conidial suspensions (1 ml of 5 x lo4 conidia ml- ‘) were pipetted onto Nucleopore filters (pore size, 0.4pm), in a Millipore filter apparatus and the liquid was gently removed by filtration. The conidia-covered filters were inserted at a 45” angle into soil in plastic pots (10.2 cm wide x 6.4 cm deep). After incubation, the filters were removed, stained with acid fuchsin in lactophenol and the conidia were observed. Germination of conidia was tested also in B solution in acid-cleaned glass Petri plates (10 ml conidium-B solution per 9 cm dia plate). In all germination experiments, clumps of more than three conidia were not counted. Duplicate samples of 100 spores in individual experiments were counted, and experiments were repeated at least twice.
60 -
o-o H
24
46
&wetted So11 Continuously Moist Soil
72
B
__
168 Hours
24
40
72
166
of Cylindrocladium scopariurn conidia (at 5 x 10“ conidia ml- ’ as inoculum) on Fig. 1. Germination four soils at 26°C. Soils (A = pH 5.5. B = pH 5.8. C = pH 6.1. D = pH 5.1) were air-dried and rewetted or maintained at near field capacity.
The response of cylindrocladium RESULTS
Germination of Cylindrocladium
conidia on and in soil
Direct observation of washed conidia of C. scoparium at 26°C indicated that peak germination (33-58x) occurred after 24 h on air-dried soils that were rewetted immediately before addition of conidia (Fig. 1). After 168 h incubation, no conidia with germ tubes were observed. Lysis of germinated conidia and germ tubes on rewetted soils began between 24 and 48 h. Lysis of non-germinated conidia was observed after 72 h, although some non-germinated conidia were observed at 168 h. Peak germination of C. scoparium conidia on continuously moist soils was lower (18-26x) than on rewetted soils (Fig. 1). Lysis of germinated and non-germinated conidia was observed at 48 and 72 h, respectively. Conidium germination on potattiextrose agar and in carbon-free inorganic salt solution (B solution) at densities of 1 x 10’ and 5 x lo4 conidia ml- ’ was 98-100x and 93-99x, respectively, after 12 h in all tests. In separate tests, percentage germination of C. scoparium conidia after 24 h on a continuously moist soil (pH 5.5), with 5 x lo4 conidia ml- ’ use,d as inoculum (the standard density used), was not appreciably different than germination when 1 x lo3 conidia ml- ’ were used as inoculum (21 and 23%, respectively). However, conidium germination after 24 h was only 3% when 1 x lo6 conidia ml-’ was used as inoculum on soil (pH 5.5). Germination in B solution at 1 x lo6 conidia ml-’ ranged from 14 to 27% (mean = 17% of
conidia to soil fungistasis
533
Table 1. Germination of Cylindrocladium floridanum and C. crotnlariae conidia on continuously moist soils at 26°C Germination (%)* C. jloridanum C. crotalariae
Hours of incubation
soil pH 5.5
soil pH 5.8
soil pH 5.5
12 24 48 72 168
24 27 15 8 0
17 23 20 12 0
21 28 21 9 0
*Assayed at an inoculum density of 5 x lo4 conidia ml-‘. Based on a count of 100 conidia for each of two replications. two replications for each of three experiments) after 12 h. Germination of washed C. scoparium conidia on
Nucleopore filters buried in three continuously-moist soils ranged from 29 to 38% after 24 h. Percentage germination decreased with time and after 168 h incubation no germinated conidia were observed. Germination on filters in no soil controls was 95-98% after 12 h. Conidia of C. jloridanum and C. crotalariae incubated on continually moist soils responded similarly to C. scoparium conidia. Peak germination of C. jloridanum conidia, on two continuously moist soils of pH 5.5 and 5.8 (27 and 23%, respectively), occurred after 24 h (Table 1). Peak conidium germination of C. crotalariae on soil pH 5.5 was 28% after 24 h.
Time after Moistening
q
Control
m
pH 5.1Soil
q
pH6.lsoll
( Days 1
Fig. 2. Germination of Cylindrocladium scoparium conidia after incubation for 16 h on washed agar disks previously exposed to volatiles from four soils. Germination was based on a count of 300 conidia (100 per disk).
DON A, ROTH and GARY
534
able to conidial germination on no-soil disks (95IOOp/, and 94-975& respectively}. In separate tests, 0.3 ml of SOJ.‘;KOH in small watch-glass chambers (2.0 cm dia) was placed in Petri dishes ~ontai~iug soil at the beginning of the period that disks were exposed to soil volatiles. When acidic volatiles, soluble in KOH, were removed from the air above the soils, germination of C. scoparium conidia was similar to germination for no-soil controls (Table 2). Presence of KOH had no effect on conidium germination in no-soil controls.
Table 2. Influence of removal of K~H-soluble volatiles on the activity of soil volatiles to Cylin~roc~u~~~~scaparium conidium germination G~rrn~nation~~ (“/b)
Soil* pH 5.1
KOH no KOH
90A 768
pH 6.1
KOH no KOH
94A
pH 5.8
KOH no KOH
95A 718
pH 5.5
KOH no KOH
44A 67B
no soil controt
KOH no KOH
96A 97A
73B
Inftuenct! of d~~~sit)e soil sofutiou on Cylindrocladium scoparium ~o~idi~rn termination In SNAD assays, germination of C. s~oparium conidia was reduced on washed agar disks exposed to one soil (pH 5.5), but was not affected appreciably on disks exposed to another soil ($-I 5.8), compared to germination on disks placed over distilled water (nosoil controls) (Table 3). Conidium germination on unwashed no-soil control disks was not appreciably different to that on washed disks. Conodium germination on disks exposed to soil pH 5.5, but not soil pH 5.8. was significantly different (P = 0.05) with time of rewetting.
* Assayed 3 days after moistening. t Based on counts of 300 conidia (100 per disk), after 16 h incubation. $ Values followed by different letters are significantly different (P = 0.05) as determined by t-test. Comparisons were made for germination values within soils. No ger~nation of washed C. scoparium conidia on soils incubated at 6°C was observed. Lysis of nongerminated conidia at 5°C was not observed until 16Xh of incubation. Further, only low germination of conidia at 6°C was observed in B solution (l-3%) and on PDA (2-8x) after 24 h. Influence of soil volatile compounds on germination
J. GRIFFIN
of
Cylindrocladium conidia Germination of C. scopurium conidia was inhibited by exposure to volatiles from four soils (Fig. 2) Germination ranged from 51 to 867;. No effect of soil moistening time was observed. Con&al germination on washed agar disks over sterile-distilled water (controls) was 9497% after 12 h. Addition of C (13.0 ng C per cnnidium as glucose) and N (65pg N ng- ‘C as NII,Cf) substrates nulhfied the inhibitory effect of soif volatiles. Germination of conidia in B solution plus C and N on disks exposed to soil volatiles was compar-
Germinability hssayed on a Cytindracladium-selective medium (Krigsvold and Griffin, 1975)at 26”C] of C. scLtparium and C. ~or~da~i#rnconidia in two artificiahy infested soils (pH 5.5 and 5.8) and C. cro~u~ariue conidia in one artificially infested soil (pW 6.1) at 26°C decreased progressively from I to 20 weeks (Fig. 3, Table 4). No germinable conidia were recovered from soils at 26°C for the 20” and 24-week assay periods. Incubation at 6°C of soils artificially infested with C. ~orjda~um, (7. scoparium and C. crotalariae conidia, resulted in no recovery of conidia after 8, 8 and 4 weeks, respectively.
Our results with C. ~orr~lar~~~, C. scopurium and C. ~or~d~~rn indicate that ~~~~~droc~ad~~rnconidia are sensitive
to
soil
fungistasis.
Similarly,
Table 3. Germination of Cylindrocladium scoparium conidia at 4 x lo6 conidia ml- ’ on washed agar disks exposed to two soil samples for 24h. using the sterile-Nucleopore-agar-diffusion method (SNAD) Germination*+ No-soil controls Non-washed agar disks (7,;)
Washed agar disks (%)
1
70
5 15
68 66
63 71 63
Time after moistening (days)
Washed agar disks soii pH 5.5 soil pH 5.8 (Xi (76) 64A 31 B 33 B
65 A 61 A 68 A
* Based on 300 conidia (100 per disk) after 5 h incubation at 26°C. T Values followed by different letters are signiticantly different (P = 0.05) as determined by Duncan’s multiple range test. Comparisons were made vertically in each column.
Morrison
The response of cylindrocladiumconidia to soil fungistasis
535
20
1
o
4
8
12
16
20
24 0
4
8
12
16
20
24
Weeks
Fig. 3. Germinability of Cylindrocladiumscoparium (A, B) and C. j?oridanum(C, D) conidia in two artificially infested soils (A, C = pH 5.5, B, D = pH 5.8) at 6 and 26°C. fungistasis. Endoconidia of five isolates of Thielavopsis basicola varied in sensitivity (O-34%) to soil fungistasis (Adams and Papavizas, 1969). Chinn and Tinline (1963, 1964) found that conidium germination of Cochliobolus sativus isolates in non-sterile soil was highly variable (O-84% germination). Germinability of Cochliobolus sativus conidia on soil was shown to be under genetic control. Soil differences also may account for the variation reported in Cylindrocladium conidial germination. Our results are the first to compare spore germination on rewetted air-dried soil to germination on continually moist soil, and demonstrate the need to use continually moist soil to determine the true level of soil fungistasis for a soil. Increased conidium germination on air-dried and rewetted soil over germination on continuously moist soils suggests that there was an increase in available C substrates in rewetted soil. Griffiths and Birch (1961) showed that peak CO* production, indicating increased microbial activity, occurred 18 h after rewetting an air-dried soil. Slightly increased conidium germination on Nucleopore membranes in soil vs conidium germination on soil may be due to condensation of water on membranes resulting Table 4. Germinability of Cylindrocladium crotalariae in a dilution of soil solution and soil microbes and conidia in an artificially infested soil (pH 6.1) at 6°C non-uniform contact between soil and the membrane and 26°C (Griffin and Pass, 1969). Ko et al. (1974) using a crude assay, indicated that Germinable conidia x lo3 g-’ soil* C. crotalariae conidia are insensitive to soil volatiles. Assay time In contrast, in our work germination of C. scoparium 6°C 26°C (weeks) conidia exposed to soil volatiles from four soils (pH 5.1-6.1) was inhibited compared to no-soil con4.1 0 8.2 trols although percentage germination of conidia 0.1 2 5.3 4 0.0 5.0 incubated directly on soil was lower than in volatile 0.0 8 4.0 assays. In comparison with germination in volatile 0.0 12 3.5 assays, the lower percentage germination of conidia 0.0 16 1.2 on soil may be due, in part, to higher volatile concen0.0 20 0.0 trations in the soil or at the soil surface than in the 0.0 24 0.0 head space above soil. In SNAD assays for soil pH 5.5, volatile fungistatic compounds alone may account *Based on C. crotalariae colonies developing on ten for the inhibition observed, although non-volatile Petri plates for each temperature per sample time. Zero compounds, detectable by this procedure, may be a time assays represent the initial germinable population. (unpublished M.S. thesis, University of Minnesota) found that C. jloridanum and C. scoparium conidia (conidium density unspecified) on non-sterile soils germinated between 1625% and 12-25x, respectively. In our study, inhibition of conidium germination probably was not a result of autoinhibition, due to the use of high conidium densities (Griffin and Ford, 1974), as comparable degrees of germination were observed on soil when 1 x lo3 conidia ml- ’ and 5 x lo4 conidia ml-’ were used as inoculum for C. scoparium. However, percentage conidium germination on soil was low when an inoculum level of 1 x lo6 conidia ml-’ was used, which indicates that the conidium density used in soil assays should be carefully controlled. Germination of Cylindrocladium conidia was C independent at low conidial densities (1 x lo3 and 5 x lo4 conidia ml-‘), but not at a high conidial density (1 x 10’ conidia ml-‘). Hwang and Ko (1974) reported that C. crotalariae conidia germinated at a high level on non-sterile soil. Possibly, clonal differences account for the large variation in sensitivity of Cylindrocladium conidia to soil
536
DON A. ROTHand
GARY
J.
GRIFFIN
contributing factor. Alternativeiy, nutrient deprivation resulting from a microbial C sink in soil may CHINN S. H. F. and TIN~IKER. D. (1963) Spore germmabrinteract with fung~s~tic compounds in lowering conility in soil as an inherent character of Coch~iobo~~4s dium germination on soil (Bristow and Lockwood, strtiuus. Phytopathology 53, 1109-l ii?. 1975). Little or no effect of soil pH 5.8 on conidium CHINN S. H. F. and TINLINER. D. (1964) Inherent germingermination was observed in SNAD assays, in comability and survival of spores of Cochliobollts S&U. parison with no-soil controls, although this soil proPhyZopar~~lo~y 54, 349 -352. duced voiatiles inhibitory to C. sco~urit~m. Diffusable GRIFFZNG. J. and PASS T. (1969) Behavior of ~~~su~i~~~i soil C compounds, which can nullify the effect of funroseurn “Sambucinum” under carbon starvation conditions in relation to survival in soil. Canadian Journal (fl’ gistatic compounds, may account for this observation Microbiology 15, 117 i 26. (Hora et ai., 1977). GRIFFING. J. and FORDR. J. (1974) Soil fungistasis: fungus Inhibitory soil volatiles, soluble in KUH, increased spore germination in soil at spore densities correspondthe exogenous C and N requirements for C. sco~~r~~?n ing to natural population ievels. Ca~~~u~i Journat of’ conidium ge~~nation. These findings are in agreel~icrob~ology 20, 751-754. ment with those Griflin et al. (1975) obtained for Fusarium
sulmi.
Conidium survival experiments corroborate results obtained from fungistasis and lysis assays. Germinable conidium populations of C. scoparium. C. joridanum and C. crotalariae in artificially infested soils at 26°C decreased progressively from 1 to 20 weeks. Germination of conidia on non-sterile soils was followed by lysis of germ tubes and germinated conidia, without formation of replacement propagules. A gradual decline in viable conidium numbers on soil was observed. Hwang and Ko (1976) found that conidia of C. ~ro~al~r~a~declined gradually during 8 months in artificially infested soil at 26°C. and suggested that thick-wagged. pigmented cells may form from germinated C. croru~~r~~e conidia. Similar structures were not observed in our experiments. Chin, and Tinline (1964) found that germinated conidia of CochlioboIus sativus lysed and disappeared from soil, but conidia of isolates which were sensitive to soil fungistasis survived longer. Failure of Cylindr~~l~~um conidia to germinate on or in soil, or survive in soil, at 6°C appears to be the result of a direct effect of temperature (Roth et al,, 1978). rather than sensitivity to soil fungistasis. ~~~~o~,~~~ge~~~rs-We thank D. T. Krigsvold for help in the preparation of figures. Contribution 375, Department of Plant Pathology and Physiology. Virginia Polytechnic Institute and State University. REFERENCES ADAMS
P. B. (1967) A buried membrane filter method for studying behavior of soil fungi. Phytoparhology 57,
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C. (1969) Survival of rootinfesting fungi in soil-X. Sensitivity of propagules of Thielauiopsis basicola to soil fungistasis in natural and alfalfa-amended soil. Phytopathology 59, 135-l 38, BRISTOWP. R. and LXXKWWD J. t. (1975) Soil fungistasis: role of the microbial nutrient sink and of fungistatic sub-
GRIFFING. J. and ROTHD. A. (1979) Nutritional aspects of soil mycostasis. fn Soil-Borne Pfant ~athoge#ls (B. Schippers and W. Gams, Ed& pp. 79-97. Academic Press, INew York. GRIFFING. J., HORA T. S. and BAKERR. (1975) Soil fungistasis: elevation of the exogenous carbon and nitrogen requirements for spore germination by fungistatic volatiles in soils. C’anirdinn ~0~~~~~ ef ~~crob~o~og~~ 21, 1468 -1475. GRIFFITI~SE. and Br~cr-r H. F. (1961) Microbioloyical changes in freshly moistened soil. Nature 189, 427. HORAT. S., BAKERR. and GRIFFING. J. (1977) Experimental evaluation of hypotheses explaining the nature of soil fungistasis. Phyto~~rho~ogy 67,.373-379. HWANGS. C. and KO W. H. (19741 Germination of Calonectsia crotahriae conidia and ascospores on soil. Mycolog& 46, 1053-1055. HWANC S. C. and KO W. H. $1976) Biology of conidia, ascospores and microsclerotia of CaEonectriu crotaluriar in soil. Phytopathology 66, 51-54. Ko W. H. and LOCKWOODJ. L. (1967) Soil fungistasis: relation to fungai spore nutrition. Phyropathologv 57.
894-901. Ko W. H.. MRA F. K. and HERLICSKAE. (1974) Isolation
and identification of a volatile fungistatic substance from alkaline soil. Phytoputhology 64, 1398-1400. KRIGSVOLDD. T. and GRIFFIN G. J. (1975) Quantitative isoiation of Cy~~ndroc~adjum crotalariae microsclerotia from naturally infested peanut and soybean field soils. Plant Disease Reporter
59. 543-546.
LINGAPPAB. T.
and LOCKWOOD J. L. (I 963) Direct assay of soils for fungistasis. Phyto~otho~ogy 53, SD.531 Loc~wooa J. L. (19773 Fungistasis in soils. Biolctyicai Review 52, l-43. ROTH D. A. and GRIFFIN G. J. (1978) The response of Cyiindrucladium conidia to soil fungistasis. Phytopathology News 12, 72-73 (Abstr.). ROTH D. A. GRIFFING. J. and GRAHAMP. J. (1978) Low temperature induces decreased germinability of C$indrocladium microsclerotia. Canadian Journal of’ Microbiology 57, 157-162.
WATSONA. G. and FORD E. J. (1972) Soil fungistasis a reappraisal’? Annual Review of Phytopoth~logy 10, 327-348.