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Influence of carbon and sources, relative carbon and nitrogen concentrations, and soil moisture on the growth in nonsterile soil of soilborne fungal antagonists

Article in Canadian Journal of Microbiology · February 2011 DOI: 10.1139/m87-109

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J. P. STACK,C. M. KENERLEY,AND R. E. PETTIT Department of Plant Pathology and Microbiology, Texas A & M Universih, College Starion, TX, U.S.A. 77843 Received July 20, ! 986 Accepted March 10, 1987

STACK,J. P., KENERLEY,C. M., and PETTIT,R. E. 1987. Influence of carbon and nitrogen sources, relative carbon and nitrogen concentrations, and soil moisture on the growth in nonsterile soil of soilborne fungal antagonists. Can. J. Microbiol. 33: 626-63 1. Three components of dispersal important to the colonization of a soil matrix by fungal parasites of sclerotia were identified and measured: the percentage of the carrier granules from which hyphae extended into soil (PGH), the mean number of hyphae extending into soil from each granule (MNH), and the mean length of the hyphae extending into soil (MLH). Factors that influence dispersal were determined for strains of Gliocladium roseum, rerricola, and Trichoderma spp. The source of carbon, the source of nitrogen, and the C:N ratio of the carrier substrate significantly (p = 0.001) affected all three components of dispersal subsequent to the placement of the carrier granules in nonsterile soil. Increased C:N ratio ( 12: 1 to 80: 1) and increased molar concentrations of both carbon and nitrogen sources (0.02 to 0.18 M maltose and 0.006 to 0.024 M arginine) gave increased PGH (17 to 82%), MNH (1 to 5 hyphae per granule), and MLH (275 to 782 pm in 24 h) for G. roseum. Similar increases in PGH (80 to 1OO%), MNH (5 to 10 hyphae per granule), and MLH (983 pm to too long and interwoven to measure after 24 h) were observed for Th. terricola. PGH and MNH were greater at high soil moistures (-0.1 and -0.33 bars matric potential; 1 bar = 100 kPa) than at low soil moisture (- 10 bars).

STACK,J. P., KENERLEY,C. M., et PETTIT,R. E. 1987. Influence of carbon and nitrogen sources, relative carbon and nitrogen concentrations, and soil moisture on the growth in nonsterile soil of soilborne fungal antagonists. Can. J. Microbiol. 33 : 626-63 1. Trois composantes de la dispersion sont importantes pour la colonisation de la matrice d'un sol par des champignons parasites des sclirotes. Identifiees et mesurees, ces composantes sont : le pourcentage de granules porteuses de germes fongiques a partir desquelles les hyphes se diveloppent dans le sol (PGH), le nombre moyen d'hyphes qui se diveloppent a partir de chaque granule (MNH) et la longueur moyenne de ces hyphes (MLH). Des souches de Gliocladium roseum, Thielavia rerricola et de Trichoderma spp. ont semi a determiner les facteurs qui influencent la dispersion. Lorsque des granules porteuses de germes furent placies dans un sol non stirilise, la source de carbone, la source d'azote et le ratio C:N du substrat des porteurs ont affect6 les trois composantes de la dispersion de fason significative (p = 0.00 1 ). Pour le G. roseum, une augmentation du ratio C:N (de 12: 1 a 80: 1) et un accroissement des concentrations molaires des sources de carbone et d'azote (le maltose, de 0,02 a 0,18 M; l'arginine, de 0,006 0,024 M) ont fait augmenter le PGH de 17 a 82%, le MNH de 1 a 5 hyphes par granule et le MLH de 275 a 782 pM en 24 h. Des augmentations similaires ont ete obsemees avec le Th. rerricola, soit de 80 a 100% pour le PGH, de 5 a 10

For personal use only. hyphes par granule pour le MNH et de 983 pM a des dimensions trop longues d'hyphes entremelis pour itre mesuries aprks 24 h pour le MLH. Dans les sols a humidite ilevie (-0.01 et -0.33 bars du potentiel matriciel; 1 bar = 100 kPa) les PGH et MNH furent plus eleves que dans les sols a faible humidit6 (- 10 bars). [Traduit par la revue]

Introduction propagule. A preliminary report of this work has been published Growth of hyphae through soil is one mechanism of active (Stack et al. lgg5). dispersal for soilborne fungi. There are several reports con- Materials and methods cerning the development and growth of mycelial strands and Strains and media rhizomorphs through artificial and natural environments (Butler The following strains were isolated from sclerotia of Aspergillus 1984; Trinci 1984; Watkinson 1984). Moisture, nutrition, and Jlavus Link. ex Fries that had been buried in nonsterile soil from C:N ratio were among the factors found to influence growth. fields of Texas: Gliocladium roseum Bain. ((31-4, Gr6), Thielavia Most of this work involved Basidiomycetes. There is little rerricola (Gilman and Abbot) Emmons (Th2, Th3), and Trichoderma available information about hyphal growth in nonsterile soil by sp. (Tri2). Gliocladium roseum Bain. (Gr1620) was obtained from a either soilborne plant pathogens or soilborne fungal antagonists sclerotium of Phymarotrichum omnivorum L. (Shear) Duggar that had been buried in a nonsterile soil from a field of Texas. These Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Kansas State Univ Lib on 12/04/13 of plant pathogens. The nature of this growth and the factors that affect growth must be understood to maximize the performance strains were selected because of their frequency of isolation from, and ability to colonize sclerotia of, either A. Jlavus or Ph. omnivorum. of biocontrol agents. Dispersal is very important in the Gliocladium roseum (Gr3), obtained from roots of alfalfa in New York epidemiology of diseases of plants. It is likely to be important in State, and the Trichoderma strains (Tham, Tharz), originally obtained the epidemiology of diseases of fungi (e.g., plant pathogens) as from soil, were included for comparative purposes. The Gliocladium well. In the case of Sporidesmium sclerotivorum, active growth and Trichoderma strains were maintained on potato dextrose agar in soil was, in part, responsible for succesful control of (PDA) (Difco Co., St. Louis, MO) at 25OC. The Thielavia strains were Sclerotinia minor in the field (Ayers and Adams 1979). maintained on 20% clarified V-8 medium (Campbells Soup Co.) at The objectives of this research were to determine (i) the 25°C. factors that influence the dispersal of soil borne antagonists and Soils (ii) whether, by manipulating these factors, dispersal can be Two soils were used in this' study. A Houston black clay (HBC) maximized. The intent is to achieve better colonization of the collected from a cotton field near Temple, TX, and a sandy-loam soil soil and increased probability of contact with the target (SSL) collected from a peanut field near Stephenville, TX. The SSL

Pr~ntedIn Canada I Imprime au Canada STACK ET AL. 627

had the following characteristics: less than 1% organic matter, pH 7.0 Growth potential experiments (determined in water), 14 pg phosphorus/g, 84 pg potassium/g, 335 Lignite granules colonized by an antagonist were dispersed over the pg magnesiumlg, 560 pg calciumlg, and a predicted low release of surface of the soil in a plastic petri dish (0.1 g lignitel5-cm dish). A nitrogen. The HBC was described previously (Kenerley and Stack nylon mesh screen (100 pm pore size; Nitex, Tetko Co., Elmsford, 1986). Moisture release curves were generated for both soils using a NY) was placed on top of the lignite. Soil was added to bury the nylon pressure plate apparatus. In growth potential experiments, soil moisture and lignite to a dept of 0.5-1.0 cm and the soil was firmed. The was adjusted gravimetrically to a percentage moisture that corresponded moisture levels of all soils were preadjusted to correspond to MPs of to a specific matric potential (MP) on the release curve. It is recognized -0.1, -0.33, - 1.O, or - 10 bars. The plates were held at 25 or 35°C that, with certain soils, rewetting a dry soil to a specific percentage for 24-72 h at which point the soil was removed to expose the nylon moisture can result in a different MP than that obtained by drying a mesh. The nylon was lifted and the lignite granules were observed at saturated soil to that same percentage moisture. Since our objective was 10 X and 40 X magnification. The following observations were made: to determine the effects of large differences in MP (-0.33 vs. - 10 bar; (i) the proportion of lignite granules from which growth occurred 1 bar = 100 kPa) rather than effects of specific MPs, the possible (either hyphal extension or sporulation); (ii) the number of hyphae discrepencies due to hysteresis were not considered. extending from each granule; and (iii) the length of hyphae extending from the lignite granule into the soil. Initial experiments indicated Lignite cultures little variability within and among replicate plates. Therefore, 20 Lignite was collected from mines near Rockdale,TX. It was ground observations per replicate plate with three replicate plates per treatment into small granules and sieved (4-mm openings) to obtain uniform were used, unless indicated otherwise. Noncolonized substrate-im- particle size. The granules were impregnated with various nutrient pregnated granules were placed in soil as controls to aid distinction of substrates by soaking in flasks (50%, by weight substrate:lignite) and hyphae of the introduced agent from hyphae of soil fungi, which might then drying for 24 h at 25OC. When dry, the lignite was again soaked in colonize and grow from the granule after placement in soil. substrate (50%, by weight) and autoclaved (20 min at 121°C). When cool, the lignite was colonized with an antagonist by the addition Growth on agar of either agar blocks from actively growing colonies or suspensions Agar disks (5 mm diameter) from the margin of actively growing of conidia (approximately lo5- 1o6 conidia/mL) from sporulating PDA cultures of G. roseum (Gr1620) or Th. terricola (Th2) were cultures. The flasks of lignite were held at 25 or 35" for 4-7 days to placed in the center of a 10-cm agar plate containing the minimal allow thorough colonization of the granules by the antagonist. 'The salts-agar (pHs 5.0 and 7.0) described above. This medium was lignite cultures were then air dried. supplemented with various carbon and nitrogen compounds in various The substrates used were thin liquid stillage, a by-product of C:N ratios. The final molarities for the carbon and nitrogen compounds sorghum fermentation (Jones et al. 1984) having a C:N ratio of were 0.02-0.05 and 0.01 -0.02 M, respectively. Radial growth was approximately 20: 1, and a minimal salts medium supplemented with determined at 48-h intervals by measuring the greatest diameter for various carbon and nitrogen sources in varying C:N ratios. The total each of five replicate plates per treatment. mass of carbon and nitrogen in the substrates were determined. For compounds (e.g., amino acids) containing both elements, the mass of Statistical analyses each element was considered in determining the total mass. For The data were tested for normality with a univariate procedure of example, when the substrate contained both maltose and arginine, the SAS (Statistical Analysis Systems, release 1985, SAS Institute, Inc., carbon in maltose and the carbon in arginine were both calculated to Cary, NC). Some data sets contained zeros and most data were not determine the total carbon mass. The C:N ratios were expressed as the normally distributed. It was determined that a log transformation of the percentage of carbon to the percentage of nitrogen relative to the mass nonzero values best approximated a normal probability distribution. of the total substrate. The ratios considered in this study were 12: 1, This was based on results of the Shapiro-Wilk test. The occurrence of For personal use only. 40: 1, and 80: 1, which represent a realistic range of C:N of the organic zeros in some data sets precluded a log transformation. To obtain matter incorporated into agricultural soils, and have been reported to accurate estimates of the true means, the procedure of Aitchison (1955) have an influence on the growth of fungi in culture. The C:N ratio was was utilized. This procedure provides minimum-variance unbiased adjusted by increasing or decreasing the amount of carbon or nitrogen estimators of the means for log normally distributed data sets sources relative to each other. containing zeros (Owen and DeRouen 1980; Pennington 1983). An The minimal salts medium contained the following (per litre): anaylsis of variance was performed (General linear models procedure, MgS04-7H20, 500 mg; CaC12.2H20, 100 mg; H3B04, 2.86 pg; SAS) using the Aitchison estimator values. MnC12.4H20, 1.41 pg; ZnS04-7H20,0.22 pg; CuSo4. 5H20, 0.08 pg; FeKEDTA, 33 mg; thiamine-HC1, 100 pg; and biotin, 10 pg. This Results medium was prepared in 0.07 M phosphate buffer at pH 5.5 and 7.0. EfSect of soil MP on growth of antagonists For experiments on solid media, the buffered minimal salts medium Lignite granules impregnated with thin liquid stillage were was supplemented with 18 g agar/L medium. colonized with strains Gr4, Th2, Tri2, and Tham. After 24 h in The minimal salts medium would not support growth of these fungi nonsterile sandy-loam soil at -0.33 or - 1.0 bar MP, hyphal without supplemental carbon and nitrogen. Concentrations of biotin extension into soil at 25 and 35°C occurred from 73 to 100% of and thiamine-HC1 (carbon- and nitrogen-containing compounds) in the granules colonized by Gr4, Th2, and Tri2 (Table 1). At the minimal salts medium were too low to significantly contribute to -

Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Kansas State Univ Lib on 12/04/13 10.0 bars MP, the percentage of granules with extended the overall C:N. hyphae (0-18%) was significantly (p = 0.001) less. Even at Substrate pH high soil moisture (- 0.33 bars), Tham grew from fewer granules To determine the effect of substrate pH upon the growth of the than the other species. antagonists, the minimal salts medium containing carbon and nitrogen The mean number of hyphae extending into soil from each sources was adjusted (1 N NaOH or 1 N HCl) to pH 5.5 or 7.0. Lignite granule increased with increasing moisture (Table 1). For granules were impregnated with the substrates as described above and strains Gr4, Th2, and Tham, there were approximately five colonized by Gr6, Gr1620, or Th2. The C-N combinations tested were times as many hyphae extending from granules at -0.33 than at maltose-arginine, galactose- arginine, fructose-arginine, fructose- nitrate, glucose-arginine, and thin liquid stillage. For each treatment, - 10.0 bars MP in the sandy-loam soil. Strain Tri2 did not grow 0.5 g lignite was added to 10 mL of nonbuffered sterile, distilled at - 10 bars. deionized water (pH 5.5), shaken well, and allowed to stand at The effect of soil moisture on the length of hyphae extending laboratory temperature. The pH was determined over time by sub- into the SSL was species dependent. There was no significant mersing the electrode into the H20 above the lignite. moisture effect (p = 0.001) for Th. terricola (Th3) at the three CAN. J. MICROBIOL. VOL. 33, 1987

TABLE1. The percentage of lignite carrier granules with growth of the biocontrol agent into soil and the mean number of hyphae per carrier granule extending into soil at two temperatures and three MPs after 24 ha

% of granules with Mean no. of growth into soil at extending three MPs hyphaelgranule at ( - bars)b three MPs (-bars)" Temperature Agent ("c) 0.33 1.0 10.0 0.33 1.0 10.0 Th. terricola (Th2) 25 35 G. roseum (Gr4) 25 35 Trichoderma sp. (Tri2) 25 35 Tr. hamatum (Tham) 25 35

"Lignite granules (1-2 mm diameter) were impregnated with thin liquid stillage, coloni~edby the indicated species of fungi, allowed toair dry, and then placed in soil. Soil moisture was adjusted gravimetrically. See text for details. bSE of the differences = 8.4. 'SE of the differences = 1.06.

TABLE2. The percentage of lignite carrier granules with growth of the biocontrol agents into soil and the mean number of hyphae per carrier granule extending into soil with two sources of carbon and three C:Nsu

Mean no. of % of granules with extending growth into soil at hyphaelgranule at three C:NS~ three C:Ns" Time Agent Substrate (h) 12: 1 40: 1 80: 1 12: 1 40: 1 80: 1 Th. terricola (Th2) M +A 24 80 100 100 5 10 10 48 90 100 100 8 10 10 For personal use only. M+NO, 24 31 96 100 2 9 10 48 47 100 100 6 10 10 G . roseum (Gr 1620) M +A 24 17 51 82 1 5 5 48 27 38 48 48 6 6 M+NO, 24 25 29 28 2 4 2 48 26 35 38 5 4 5

"Lignite granules were impregnated with minimal salts medium containing maltose and arginine (M+ A) or maltose and potassium nitrate (M+N03)as the primary sources of carbon and nitrogen at C:Ns of 12: 1, 40: 1, and 80: 1. After colonization by Th2 or Gr1620, the granules were air dried and placed in soil at -0.33 bars MP and 35°C. bSE of the differences = 5.51. "SE of the differences = 0.64.

levels of MP tested. However, G.roseum (Gr3), Tr. hamatum stillage was 5.3 whether or not the granules were colonized by (Tharn), and Tr. harzianum (Tharz) had less (p = 0.001) Gr6. The granules impregnated with fructose-nitrate and

Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Kansas State Univ Lib on 12/04/13 extension into soil at - 10.0 than at -0.33 bars MP. This effect glucose-arginine that were colonized by Th2 had pH 5.7. All was more pronounced at 25 than at 35°C. Variability with other combinations of C-N source and isolate (Th2 or Gr l620), respect to hyphal extension into soil was observed among the regardless of initial substrate pH (5.5 or 7.0), had pH 6.0-6.2. three strains of Trichoderma. The Tri-2 strain grew further than The efSect of carbon and nitrogen source and C:N ratio on the other two strains at all moisture levels tested. The effects of growth in nonsterile soil soil moisture and soil temperature upon the three growth The source of carbon, the source of nitrogen, and the relative parameters of antagonists in the SSL were also observed in the concentrations of carbon and nitrogen affected the growth of the HBC. antagonists in a nonsterile soil. The nature of the effect was Substrate pH manifested in three ways: the percentage of carrier granules with There was essentially no difference among the pH values growth of the antagonist (Table 2), the number of hyphae recorded at 10 min or 5 h after immersing the lignite granules in growing from individual granules (Table 2), and the length of water. The pH of lignite granules impregnated with thin liquid the hyphae extending into soil from the carrier granule (Table STACK ET AL. 629

TABL.E 3. The mean length of hyphae extending into soil of hyphae at C:N of 80:l was two- (maltose-arginine) and from the carrier granules impregnated with various sources five-fold (maltose-nitrate) higher than at 12: 1. For Grl620 at 24 and C:Ns after 24 ha h, the number of hyphae per granule at the C:N of 80:l was fivefold higher than at the C:N of 12: 1 with maltose-arginine, Hyphal length (km) but there was no increase when the C:N ratio of maltose-nitrate at three C:Ns was altered. These differences were significant at p = 0.001. At a C:N of 80:1, there were almost twice as many hyphae Agent Substrate 12: 1 40: 1 80: 1 extending from the granules colonized with Th. terricola as Th, terricola (Th2) M +A 983 734 OG those colonized with G. roseum. M+N03 166 568 OG In most experiments, the hyphal growth by Th2 from G.roseum (Gr 1620) M +A 275 697 782 granules impregnated with maltose-arginine at 80: 1 was too M+N03 442 434 436 extensive (long and interwoven) to accurately determine hyphal length. When we removed the nylon screen from these plates, a 'lignite granules were impregnated with minimal salts medium containing maltose and arginine (M+A) or maltose and potassium nitrate (M+N03)as layer of soil would often remain attached to the screen owing to the primary sources of carbon and nitrogen at C:Ns of 12: 1,40: 1, and 80: 1. the extensive hyphal network throughout the soil. Similar After colonization by Th2 or (31620, the granules were air dried and placed growth (interwoven hyphae too long to measure) was observed in soil at -0.33 bars MP and 35°C. The SE of the differences = 116. with maltose-nitrate at 80: 1, though less consistently. For Th2 and Gr1620 on maltose-nitrate and Gr1620 on maltose- 3). The values in Tables 2 and 3 are the results of a single arginine, the length of hyphal extension into soil increased as experiment conducted in the HBC soil. The experiment was the C:N was increased from 12: 1 to 80: 1. For Th2 with repeated twice with comparable results. Similar experiments maltose-nitrate, the average length increased from 166 mm at were conducted in the SSL soil and comparable results were the C:N of 12: 1 to hyphae too long to measure at the C:N of obtained. The magnitude of the values varied among experi- 80:l. For Gr1620 with maltose-arginine, there was almost a ments, but the nature of the growth response remained consistent threefold increase in the average hyphal length. With maltose- (e.g., increased C:N from 12: 1 to 80: 1 resulted in an increased nitrate, there was no significant difference in the average length number of hyphae per carrier granule in each experiment). We of hyphae at the three C:Ns tested. The effects of substrate, also observed this effect of C:N on the growth response of Th2 isolate, and C:N ratio, as well as their interactions, were and Gr1620 with other sources of carbon and nitrogen, e.g., statistically significant (p = 0.00 1). galactose-ammonium and galactose-nitrate. In initial experi- Experiments were conducted to determine the effect upon ments, we observed that after 48 h in nonsterile soils, HBC and Gr1620 of decreasing the C:N by holding the concentration of SSL, at 25°C and - 1.0 or -0.33 bars MP, the percentage of carbon constant (at the level for C:N = 80: 1) and increasing the granules with hyphal growth and the length of Th. terricola nitrogen concentration (Table 4). As expected from earlier (Th2) hyphae extending into soil were greater with maltose or experiments, increased carbon concentration resulted in a galactose (primary carbon source) in combination with arginine greater number of granules with hyphal growth (sevenfold (primary nitrogen source) than with fructose and arginine. increase). In addition, Gr 1620 was sporulating from 100% of Galactose in combination with arginine gave a greater growth the granules. Increasing both the carbon and nitrogen con- For personal use only. response by Th2 than galactose with KN03. centrations such that the C:N remained 12: 1 resulted in a In one experiment with Gr 1620, the growth response from a comparable increase in the proportion of granules with hyphal defined synthetic medium (minimal salts with galactose- growth. Unlike the treatment where only the carbon con- ammonium) was much greater than growth from an undefined centration was increased, however, Gr1620 did not sporulate on natural substrate (thin liquid stillage). At 25°C and -0.33 bars the granules. There were more hyphae per granules and the MP there were 100 and 13% granules with hyphal extension, and mean length of hyphae was greater where the nitrogen con- nine and two hyphae per granule for the synthetic and natural centration was also increased (significant at p = 0.05). substrates, respectively. Similar results were observed at 25°C with - 1 .O bar MP and at 35°C with -0.33 and - 1.O bars MP. Effect of carbon and nitrogen source and the C:N ratio on As the ratio of C:N was increased from 12: 1 to 80: 1, the growth on agar percentage of granules with hyphae extending into the soil Minimal salts - agar amended with carbon and nitrogen increased for both G. roseum and Th. terricola (Table 2). This sources in various ratios was seeded with the agents to identify was true at 24 and 48 h whether the nitrogen source was arginine optimal substrates and substrate combinations for use in soil or nitrate, with the exception of G. roseum at 24 h with nitrate. studies. Differences in preference for carbon and nitrogen Thielavia terricola (Th2) grew from a much higher percentage source were observed on agar for both agents (Table 5). Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Kansas State Univ Lib on 12/04/13 of the carrier granules than G. roseum (Gr1620) under most However, the growth response on agar did not correlate well conditions of substrate. This was statistically significant (p = with the growth observed in nonsterile soil (Tables 2 and 3). On 0.001). agar, galactose was the poorest source of carbon for growth of The increase in C:N from 12: 1 to 80: 1 was equivalent to an G. roseum, yet in soil it was one of the best, yielding 100% of increase in the molarity of the carbon source maltose from 0.02 the carrier granules with hyphal extension and seven hyphae per to 0.18 M, while the molarities of the nitrogen sources carrier granule at -0.33 bars MP and 35°C. Similar results were arginine and nitrate remained constant at 0.006 and 0.024 M, obtained at 25°C. In contrast to the observations in nonsterile respectively. soil (Table 6), G. roseum grew to greater colony diameters than Similar results were observed when the number of hyphae per Th. terricola in equal time periods at the three C:Ns tested granule was considered. As the C:N was increased from 12: 1 to (Table 6). This was true whether arginine or nitrate was the 80:1, the number of hyphae extending from each granule source of nitrogen. Growth on agar did not predict relative increased for both agents (Table 2). For Th2 at 24 h, the number performance in soil of the different antagonist strains nor did it CAN. J. MICROBIOL. VOL. 33, 1987 TABLE4. The effect of relative carbon and nitrogen concentrations on the growth of G. roseum (Grl620) in nonsterile soil from carrier granules impregnated with different concentrations of maltose and arginine"

Molarity X % granules with: No. Mean length C:N Maltose Arginine hyphae sporulation no growth hyphaelgranule of hyphae (pm)

"Lignite granules were impregnated with minimal salts medium containing maltose and arginine as the primary sources of carbon and nitrogen at C:Ns of 80: 1 and 12:l. Two concentrations of carbon and nitrogen were used to achieve a C:N of 12: 1. After colonization by G. roseum (Gr1620). the granules were air dried and placed in soil at -0.33 bar MP and 35°C. bNot detennined owing to high proportion of granules with no growth.

TABLE5. Mean colony diameter after 7 days growth on minimal salts - agar supplemented with various sources of carbon (0.02 M) and nitrogen (0.01-0.02 M)

Colony diameter (mm)" with various carbon sources Nitrogen Agent source Glucose Sucrose Maltose Fructose Galactose Th. terricola (Th2) Arginine KN03 (NH412S04 Glutamic acid G. roseum (Gr 1620) Arginine KN03 (NH4)2S04 Glutamic acid

"Mean of five replicate plates.

TABLE6. Colony diameter after 10 days on a defined medium with The sources of carbon and nitrogen significantly affected the maltose and nitrate or arginine at three C:Ns growth of antagonist species in nonsterile soil and on agar. An attempt was made to utilize the growth response on agar as a Colony diameter preliminary screen to identify the optimum sources of carbon (mm)a at various and nitrogen for each antagonist and to predict their relative For personal use only. C:Ns performance in natural soil. However, there was little correlation of growth response on agar to the growth response in soil. Agent Carbon Nitrogen 80: 1 40: 1 12: 1 Galactose, a good source of carbon for hyphal elongation of G. G. roseum (Gr 1620) Maltose NO3 55 54 49 roseum in soil, was the poorest source for growth on agar. It also Maltose Arginine 57 57 56 was evident that the ability to colonize the substrate-impregnated Th. terricola (Th2) Maltose NO3 19 18 16 carrier granules in the preparation stage was not an indication of Maltose Arginine 35 34 25 performance in soil. For certain antagonist-substrate combina- tions, the antagonist sporulated profusely subsequent to addition "Mean of five replicate plates per treatment. to soil, but did not grow from the granule into the soil. This was true with isolates of Trichoderma and Gliocladium . Although the substrate was good for biomass production, it was not predict the relative performance in soil of different carbon and suitable for achieving the desired growth response (i.e., hyphal nitrogen sources or ratios. extension). The initial concentrations of carbon and nitrogen were Discussion

Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Kansas State Univ Lib on 12/04/13 consistent with the requirements of fungi for growth and Colonization of a soil matrix by an introduced agent would sporulation in culture (Griffin 198 1). Increasing the carbon comprise at least three components of hyphal dispersal: the concentration (0.02 to 0.18 M) such that the C:N ratio of proportion of the agent-carrier population from which hyphae the carrier substrate (maltose-arginine or maltose-nitrate) emerge, the number of hyphae extending into soil from each increased from 12: 1 to 80: 1 resulted in enhanced dispersal in agent-carrier unit, and the length of hyphal extension into soil soil, as indicated by a greater percentage of carrier granules from the agent-carrier unit. Also important, but not considered from which growth occurred, a greater number of hyphae in this study, is the hyphal branching pattern in soil. To extending from each carrier granule, and an increased length of adversely affect plant pathogen propagules in soil, an antagonist the hyphae extending into the soil. By simultaneously increasing must first reach the target propagules. In this study, several the nitrogen source concentration (0.006 to 0.042 M) such that parasites of sclerotia of A. jlavus and Ph. omnivorum were the C:N ratio remained 12: 1,.a further increase in the number of evaluated for their ability to colonize soil relative to the above hyphae per carrier granule and a greater mean length of hyphae criteria. Also, an attempt was made to determine factors that extending into soil resulted. Even after subsequent experiments, affect the three components of dispersal. it was difficult to determine whether the effects were a result of STACK ET AL. 63 1

an increase in molar concentration of the carbon or nitrogen DAN 4048-G-55-2065-00, and the Texas Agricultural Experi- source, or a result of a change in the C:N ratio. ment Station. We thank Curt Cocking, Jeanne Cortese, and There are several reports on the effects of the sources and Malinda McMurray for technical assistance and Trey Richard- ratios of carbon and nitrogen on the growth rate and habit of son, Institute of Statistics, Texas A & M University, for fungi (Trinci 1984; Thompson 1984; Watkinson 1984). Most assistance with the statistical analyses. studies were conducted on artificial media; others were con- ducted on natural substrates such as wood. In these studies, the C:N ratio affected the branching pattern and the rate of AITCHISON,J. 1955. On the distribution of a positive random variable having a discrete probability mass at the origin. J. Am. Stat. Assoc. elongation of hyphae. In trying to encourage a more thorough 50: 901-908. colonization of soil, these are the precise growth attributes we ALEXANDER,M. 197 1. Microbial ecology. John Wiley & Sons, New want to effect. This should lead to a more thorough colonization York. of the soil and increase the probability of contact with the target AYERS,W. A., and ADAMS,P. B. 1979. Mycoparasitism of sclerotia of propagule. Sclerotinia and Sclerotium species by ~ioridesmiumsclerotivorum. Soil moisture also significantly affected dispersal of the Can. J. Microbiol. 25: 17-23. soilborne fungi. By determining the effects of soil moisture on 198 1. Mycoparasitism and its application to biological control dispersal, we will be in a better position to integrate application of plant diseases. Beltsville Symp. Agric. Res. 5: 91- 103. of a biocontrol agent with management practices, such as BAKER,K. F., and COOK,R. J. 1974. Biological control of plant irrigation and (or) to take advantage of natural rainfall. The pathogens. W. H. Freeman and Company, San Francisco, CA. BUTLER,G. M. 1984. Colony ontogeny in basidiomycetes. In The timing of application of the agent with respect to the environment ecology and physiology of the fungal mycelium. Edited by D. H. and the crop will be critical to maximize the agents' performance. Jennings and A. D. M. Rayner. Cambridge University Press, There have been few cases of effective control of soilborne Cambridge. pp. 53-71. plant pathogens by the introduction into soil of antagonists to COOK,R. J., and BAKER,K. F. 1983. Introduction of antagonists for those pathogens. Many reasons have been suggested to explain biological control. In The nature and practice of biological control of this lack of success. Some feel that introducing an alien plant pathogens. American Phytopathological Society Press, St. organism, e.g., antagonist, is predisposed to failure because the Paul, MN. pp. 282-3 11. introduced organism is inferior to the native soil microflora ELAD,Y., HADER,Y., CHET,I., and HENIS,Y. 1982. Prevention with (Alexander 197 1; Garrett 1956). It has also been stated that Trichoderma harzianum Rifai aggr., of reinfestation by Sclerotium reintroduction of indigenous antagonists is predisposed to rolfsii Sacc. and Rhizoctonia solani Kuhn of soil fumigated with failure because a give soil will support only a given population methyl bromide, and improvement of disease control in tomatoes and . Crop Prot. 1: 199-21 1. (Baker and Cook 1974; Garrett 1956). However, their con- GARRETT,S. D. 1956. Biology of root-infecting fungi. Cambridge clusions are based on the behavior of microorganisms in natural University Press, Cambridge. ecosystems. There is great potential for manipulation of GRIFFIN,D. H. 198 1. Fungal physiology. John Wiley and Sons, Inc., agroecosystems to establish conditions that will allow the New York, NY. introduced biocontrol agent to overcome natural barriers to JONES,R. W., PETTIT,R. E., and TABER,R. A. 1984. Lignite and proliferation. Site modification has been achieved by solarization stillage: Carrier and substrate for application of fungal biocontrol agents to soil. Phytopathology , 74: 1167- 1 170.

For personal use only. (Katan 1980), fumigation (Elad et al. 1982), and irrigation. Modification at the microsite level could be accomplished by KATAN,J. 1980. Solar pasteurization of soils for disease control: status use of specific carrier substrates that elicit desired agent and prospects. Plant Dis. 64: 450-454. responses. KENERLEY,C. M., and STACK,J. P. 1986. Influence of assessment methods on selection of potential fungal antagonists of the sclero- It has been stated that, before we can develop effective tium-forming Phymatotrichum omnivorum Can. J. Micro- biological control practices, we must first learn as much as biol. 33: This issue. possible about the life history of the plant pathogen (Baker and MANOCHA,M. S. 1981. Host specificity and mechanism of resistance Cook 1974; Cook and Baker 1983; Doupnik 1984'). It can be in a mycoparasitic system. Physiol. Plant Pathol. 18: 257-265. argued that we must learn equally as much about the antagonist OWEN,W. J., and DEROUEN,T. A. 1980. Estimation of the mean (Ayers and Adams 1981; Cook and Baker 1983; Kenerley and for log normal data containing zeros and left-censored values, Stack 1986). Similarities between mycoparasites and plant with applications to the measurement of worker exposure to air pathogens have been reported with respect to mechanisms of contaminants. Biornetrics, 36: 707-7 19. pathogenesis, e.g., prepenetration and penetration activities PENNINGTON,M. 1983. Efficient estimators of abundance for fish and (Manocha 1981). The analogy could be extended to include plankton surveys. Biornetrics, 39: 28 1-286. STACK,J. P., KENERLEY,C. M., and PETTIT,R. E. 1985. Growth other factors which make a pathogen effective, e.g., production potential in soil of hyperparasites of sclerotia. Phytopathology, 75: Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Kansas State Univ Lib on 12/04/13 and dispersal of inoculum. This study provides information on 1 344- 1 345. dispersal of soilborne fungi and factors that influence dispersal THOMPSON,W. 1984. Distribution, development and functioning of in soil. mycelial cord systems of decomposer basidiomycetes of the deci- duous woodland floor. In The ecology and physiology of the fungal Acknowledgements mycelium. Edited by D. H. Jennings and A. D. M. Rayner. This study was supported by the United States Agency for Cambridge University Press, Cambridge. pp. 185-2 14. International Development, Department of State, grant no. TRINCI,A. P. J. 1984. Regulation of hyphal branching and hyphal orientation. In The ecology and physiology of the fungal mycelium. Edited by D. H. Jennings and A. D. M. Rayner. Cambridge University Press, Cambridge. pp. 23-52. '~oupnik,B. 1984. Sorghum root and stalk rots, a critical review: WATKINSON,S. C. 1984. Morphogenesis of the Serpula lacrimans Proceedings of the Consultative Group Discussion on Research Needs colony in relation to its function in nature. In The ecology and and Strategies for Control of Sorghum Root and Stalk Rot Diseases. physiology of the fungal mycelium. Edited by D. H. Jennings and A. International Crops Research Institute for the Semi-Arid Tropics, D. M. Rayner. Cambridge University Press, Cambridge. pp. Patancheru, A.P. 502 324, . 165-184. This article has been cited by:

1. G. R. Knudsen, J. P. Stack, S. O. Schuhmann, K. Orr, C. LaPaglia. 2006. Individual-Based Approach to Modeling Hyphal Growth of a Biocontrol Fungus in Soil. Phytopathology 96:10, 1108-1115. [CrossRef] 2. Y.S. Bae, Guy R. Knudsen. 2005. Soil microbial biomass influence on growth and biocontrol efficacy of Trichoderma harzianum. Biological Control 32:2, 236-242. [CrossRef] 3. J. P. Clarkson, A. Mead, T. Payne, J. M. Whipps. 2004. Effect of environmental factors and Sclerotium cepivorum isolate on sclerotial degradation and biological control of white rot by Trichoderma. Plant Pathology 53:3, 353-362. [CrossRef] 4. John J. Classen, Cady R. Engler, Charles M. Kenerley, A. Dale Whittaker. 2000. A logistic model of subsurface fungal growth with application to bioremediation. Journal of Environmental Science and Health, Part A 35:4, 465-488. [CrossRef] 5. Yong-Ha Park, James P. Stack, Charles M. Kenerley. 1991. Production of gliotoxin by Gliocladium virens as a function of source and concentration of carbon and nitrogen. Mycological Research 95:10, 1242-1248. [CrossRef] 6. C. KEEL, M. MAURHOFER, T. OBERHÄNSLI, C. VOISARD, D. HAAS, G. DEFAGORole of 2,4- Diacetylphloroglucinol in the Suppression of Take-All of Wheat by a Strain of Pseudomonas Fluorescens 23, 335-338. [CrossRef] For personal use only. Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Kansas State Univ Lib on 12/04/13

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