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II IIII II III III I II..I.II.n I'ID'II III THESIS University This is to certify that the thesis entitled ETIOLOGY AND EPIDEMIOLOGY 0F PYTHIUM ULTIMUM PREEMERGENCE DAMPING-OFF 0F SOYBEAN AND OF FUSARIUM SPECIES AS SECONDARY PATHOGENS presented by ROBERT L. SCHLUB has been accepted towards fulfillment of the requirements for Ph. D. degree in Plant Pathology Major professor 2%sz Date Oct. 5, 1979 0-7 639 (I'm FIN£§: 25¢ P" w 90" 1t- RETUMIIIS LIBRARY MTERIAL§= Place In book atom to name charge fro. circulation records ETIOLOGY AND EPIDEMIOLOGY OF PYTHIUM ULTIMUM PREEMERGENCE DAMPING-OFF OF SOYBEAN AND OF FUSARIUM SPECIES AS SECONDARY PATHOGENS By Robert L. Schlub A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1979 ABSTRACT ETIOLOGY AND EPIDEMIOLOGY OF PYTHIUM ULTIMUM PREEMERGENCE DAMPING-OFF OF SOYBEAN AND OF FUSARIUM SPECIES AS SECONDARY PATHOGENS By Robert L. Schlub This study was undertaken to find the cause of preemergence damping-off of soybean seedlings in sandy soils of Southwest Michigan and to determine the environmental conditions favoring the disease. Several areas of research were undertaken. A recording soil moisture meter, de- scribed in Part I, was designed and built so that soil moisture data from the field could be used as a variable in a multiple regression equation to predict seedling emergence in the field. In Part II, a lab- oratory method was developed so that a large number of potential soil- borne pathogens could be screened for pathogenicity. Species of Fusari- um are known to cause root rot of soybean and are frequently isolated from diseased plants; therefore, they were intensely studied in Part III. The cause of the preemergence disease was found to be due to Pythium ultimum; its ecology and epidemiology are researched in Part IV. I. The soil moisture meter consisted of a 741 IC amplifier and a . 0-1 mA meter to measure the voltage drop from a 741 IC oscillator caused by the impedance of gypsum soil moisture blocks. The blocks' impedance, measured in ohms, changes with the matric potential of the soil. The circuit design eliminated the need for manual balancing and incorporated zener diodes to insure stable readings. When connected with a 0-1 mA Robert L. Schlub time-share two input recorder and a relay system, as many as 13 soil moisture blocks can be read by the same instrument. The meter's readings varied only i % of full scale (:25 ohm) over three two—week field trials. 11. The pathogenicity of several fungal isolates obtained from soil and diseased soybean plants was evaluated in 2.5 X 12 cm polysty- rene tubes. Two soybeans were placed on a 17 mm diameter agar disk of the test fungus which in turn rested on a 4—cm deep layer of fine white sand. The side drainage holes were sealed with adhesive tape and the bottom drainage hole was covered with nylon netting to retain the sand. Soybean seeds were covered with a 4-cm layer of a mixture of vermiculite and perlite (7:3, v/v). The tubes were watered daily and incubated at 25 C for 11 days under fluorescent lights. This method gave good lesion development wi th Pythium, Phytophthora, Thielaviopsis, Rhizoctonia, and Fusarium isolates. III. Fusarium spp. colonized nonsterilized dead plant tissue pieces such as wheat straws, corn stalks, soybean pods, soybean stems, and sterilized soybean stems. The colonization of the tissue resulted in an increase in the number of propagules in the soil within a 3-mm distance from the tissue surface. The number of Fusarium propagules was higher in the soil adhering to tissue pieces placed near the soil surface than in those buried 8 or 15 cm deep. Fusarium spp. were also found to be able to colonize soybean seeds in dry soil. Emergence of "Hark" soy- bean seedlings from seeds planted 3 cm deep in natural field soil de- creased with the number of days soil was held at -15 bars matric poten- tial before watering to -O.2 bar. Robert L. Schlub IV. Lesan, a fungicide which controls Pythiaceous fungi, was added to field soil under conditions favorable for preemergence damping- off of soybean. This treatment restored emergence, thereby indicating that Pythium spp. were the primary cause of the disease. When Fusarium spp. were added to soil containing Pythium, disease was no more severe than with Pythium alone. From laboratory studies, it was found that emergence was lowest in wet soil (-18 m bar) whether artificially or naturally infested with P. uZtimum. Poor emergence in wet, infested soil was verified using multiple regression analysis of disease inci- dence and soil moisture data collected in the field. Regression analy- sis also verified the minor importance of temperature in the range of 20 to 28 C in disease development, which also agreed with laboratory re- sults. To my parents 11' ACKNOWLEDGMENTS To Dr. John L. Lockwood, I express gratitude for the faith he has shown in my ability to complete this degree, and for the constant attention he has given me in the last few months. To the members of my guidance committee--Drs. L. Copeland, M. Lacy, and G. Safir--I extend my thanks for their suggestions and for their evaluation of the manusCript. I thank Dr. R. Kunze for his assistance in using and calibrating soil moisture blocks. I would like to extend my thanks to Dr. C. Cress and Mr. Scott Eisensmith for their assistance in statistical evaluation of data, and to Mr. Scott Eisensmith for guidance in using computer programs. I also extend my appreciation to Mr. James Maine and Mr. John Houchlei who assisted me in the construction of the soil moisture meter. I would like to thank Mr. and Mrs. Richard Bothamley for pro- viding the necessary field plots and for their generosity and hospitality. I am most appreciative of my wife, Joanne Di Lucca Schlub, for her constant encouragement and for providing valuable assistance in con- ducting experiments and preparing this manuscript. TABLE OF CONTENTS Page LIST OF TABLES ........................... vi LIST OF FIGURES ........................... viii GENERAL INTRODUCTION ...................... '. . 1 PART I PORTABLE RECORDER FOR THE CONTINUOUS MONITORING OF SOIL MOISTURE RESISTANCE BLOCKS INTRODUCTION . . . . . ....................... 4 Description of recording soil moisture meter .......... 6 Description of the moisture meter circuit ............ 8 Performance ........................... 10 PART II A LABORATORY METHOD FOR SCREENING POTENTIAL SOIL-BORNE PATHOGENS OF SOYBEAN INTRODUCTION ............................ 14 MATERIALS AND METHODS ........................ 16 RESULTS ............................... 19 DISCUSSION ............................. 25 PART III COLONIZATION OF PLANT TISSUE AND SOYBEAN SEEDS BY FUSARIUM SPECIES IN NATURAL AND ARTIFICIALLY INFESTED SOIL INTRODUCTION ............................ 27 MATERIALS AND METHODS ............... . ........ 31 Estimation of Fusarium population ................ 31 Propylene oxide treatment of soil, seeds and dry plant tissue. . 32 Pathogenicity test for Fusarium isolates ............ 33 Soil characteristics and matric potential determinations . . . . 33 Saprophytic survival in nonsterile tissue in the field ..... 35 Influence of soybean residue on the bulk soil population . . . . 35 iv Page Saprophytic colonization of disinfected tissue ......... 36 Preemergence rotting of soybean seeds by Fusarium. . . ..... 37 RESULTS ............................... 38 Saprophytic survival in soil and nonsterile tissue ....... 38 Saprophytic colonization of disinfected tissue ......... 40 Preemergence rotting of soybean seeds by Fusarium ........ 45 DISCUSSION ............................. 49 PART IV ECOLOGY AND EPIDEMIOLOGY OF PYTHIUM ULTIMUM PREEMERGENCE DAMPING-OFF OF SOYBEANS INTRODUCTION ............................ 53 MATERIALS AND METHODS ........................ 55 Laboratory experiments ...................... 55 Collection of field data ..................... 57 Soil moisture block calibration and field use ........... 57 Multiple regression analysis ................... 58 RESULTS ............................... 59 Influence of inoculum and matric potential on emergence ...... 59 Influence of temperature on emergence ............... 63 Investigating a possible Fusarium-Pythium interaction ....... 63 Multiple regression analysis using field data ........... 66 DISCUSSION ............................. 72 LITERATURE CITED .......................... 77 Table LIST OF TABLES Page PART II Severity of soybean root rot caused by five fungal isolates used to infest five different planting media in plastic tubes ....... . . . . . . . . . . . . . . . 20 Severity of soybean seedling rot caused by six fungal isolates used to infest five different planting media in plastic tubes . . . ....... . ..... . ..... 21 Severity of root rot of soybeans planted in plastic tubes caused by five fungal isolates under four lighting and watering schedules . . . . . . . . . . . . . . . . . . . 22 Severity of seedling rot of soybeans planted in plastic tubes caused by seven fungal isolates under four lighting and watering schedules ............... 24 PART III Population density of Fusarium in a sandy-loam soil and in mature root tissue collected at four different times. . . 39 Population density of Fusarium in a sandy-loam soil and in mature root tissue pieces buried at three different soil depths for 9 months ............. . . . . . 41 Population density of Fusarium in a sandy-loam field soil and soil which adhered to plant tissue pieces after they were buried for 9 months in the field ..... . ...... 42 Population density of Fusarium in soil which adhered to disinfected soybean stem pieces buried in the soil at 3 matric potentials and in soil not associated with stem pieces at -90 bars (control) sampled at three time periods. . . . . . . . . . . . . . . . . . . . . ...... 43 Emergence of soybean seeds planted in a sandy-loam field soil treated with Lesan or with Lesan and benomyl and held for 6 days at 5 different matric potentials before watering to -O. 24 bar. . . . . . ............. 47 vi Table 10. 11. 12. 13. 14. 15. 16. PART IV The effect of Pythium uZtimum inoculum density in soil (-0. 18 bar matric potential) on emergence of soybean seedlings at 28 C .................... The influence of matric potential on soybean emergence in propylene-oxide treated soil with and without Pythium ultimum (7 propagules/g) at 28 C ......... The influence of soil matric potential values on soybean emergence in a naturally Pythium uZtimum infested sandy- loam soil (500 propagules/g) at 28 C ........ . . . . Effect of temperature on the emergence of soybeans in disinfested soil infested with Pythium ultimum (7 propagules/g) and in non-infested soil .......... Percentage of soybean seedling tissue pieces from which Pythium and Fusarium spp. were isolated when planted in natural soil (300 ppg Pythium and 5,000 ppg Fusarium) at 28 C for 1,3 and 6 days ............ . Actual emergence of soybeans planted over the summer, 1978, compared with predicted values based on a multiple regression equation ................... A partial list of independent variables which were eliminated from the regression equation due to their low level of significance (>10%) ............. vii Page 60 61 62 64 65 69 71 LIST OF FIGURES Figure Page PART I 1. A block diagram of the recording soil moisture meter ..... 7 2. Circuit diagram of a single channel used in the recording soil moisture meter. (8) soil moisture block; (C1)(C2)(C3) 0.1 pF; (C4) 0.47 uF; (C5) 50 pF; (D1)(DZ) IN58A; (03) IN34A; (IC1)(IC2) 741 op. amp.; (M) 0-1 mA d.c. in recorder; R1) 10 k; (R2)(R3)(R4) 1 k; (R5) 100 k; (VRl) 50 k; (VRZ) 10 k; (VR3)(VR4) 50 k ................. 9 3. Mounting of recording soil moisture meter and tem- perature recorder in styrofoam ice chest .......... 11 PART II 4. Cross-section of a polystyrene tube used for assessing pathogenicity of fungal isolates to soybean. Various media were placed above and below the seed and inoculum agar disk .......................... 18 PART III 5. Soil moisture-matric potential curve of a sandy-loam soil obtained by desorption with a pressure plate apparatus .......................... 34 6. Emergence of soybean seedlings from seeds sown in a sandy- loam soil held at -15 bars for 0, 2, 4 and 6 days before watering to -0.24 bar. Values are based on three consecu- tive plantings of 20 seeds per treatment in the same soil. Means followed by different letters are significantly different from each other using Tuke 's w rocedure (E_= 0.05) on transformed data: 7 x + 5.5 ......... 46 7. Emergence of soybean seeds planted over the summer with correSponding data on rainfall, soil temperature and soil moisture. The soil, a sandy-loam, was naturally infested with Pythium ultimum. The emergence data were recorded 2 to 3 weeks aftér planting and are shown on the figure on the first day of planting .................. 67 viii GENERAL INTRODUCTION The purpose of this research was to determine the cause of pre- emergence damping-off of soybean seedlings in sandy soils of Southwest Michigan and to determine the environmental conditions favoring disease development. The disease has been known to occur in these areas for at least the past 10 years. The primary symptom is preemergence rotting of the germinated seedlings. Infected seedlings normally have swollen hypocotyls and lesions at the junction of the hypocotyl and primary root. Other symptoms include a curling growth habit and reddish to brown le- sions on the hypocotyl and cotyledons. Replanting in an area of poor emergence often results in good emergence, indicating that environmental factors are important in disease development. One of the most important environmental parameters is soil moisture. This was measured in the field with a soil moisture meter, whose design, construction, and opera- tion are outlined in Part I. Soil moisture, soil temperature and rain- fall data were collected in the field and used to form a predictive re- gression equation of the disease which is contained in Part IV. The fungi most frequently isolated from plant lesions were Fusarium spp. (80-95%) and Pythium spp. (10-60%). Fusarium species also were frequently isolated from soil, plant residues and lesions from diseased plants. In order to determine which of the Fusarium isolates were pathogenic, a method was developed to test a large number of isolates for pathogenicity (Part I). Since Fusarium*oxysporum, which is known to cause root rot of soybean, was frequently isolated from diseased plants, its ecology was intensely studied in Part III. Later in the course of my research, by using selective fungicides, Pythium uZtimum was determined to be the cause of the disease. Its pathogenic capabili- ties were studied in Part IV. PART I PORTABLE RECORDER FOR THE CONTINUOUS MONITORING OF SOIL MOISTURE RESISTANCE BLOCKS INTRODUCTION The degree to which water in the soil is available to the host and soil-borne pathogens often determines disease development; therefore, soil moisture should be recorded with other biological and environmental data in epidemiological studies. water availability is expressed by its potential to do work relative to the work capacity of pure free water and can be expressed as free energy (joules/kg) or as pressure (1 bar 100 joules/kg). "Since water is held in the soil by forces of adsorption, cohesion, and solution, soil water is not usually capable of doing as much work as pure free water, hence the potential is negative" (119). The soil water potential is the sum of the osmotic, matric, gravi- tational and pressure potentials. In soil, the osmotic potential is de- termined by the amount of dissolved salts. The osmotic potential is usually low and is not a major component of the soil's water potential unless the soil is very wet or heavily fertilized. Matric potential is determined by the soil's adsorptive and capillary properties. The matric potential of the soil can easily be determined by finding its percentage of moisture values as the x and y axes (Figure 5). The moisture-matric potential curve can be determined by using a pressure plate apparatus (96). Pressure is applied to the soil in the apparatus; when equilibri- um is reached, the soil matric potential will equal the pressure applied and the percent moisture value can then be determined. It is often desirable to measure soil moisture continually, and recently several automatic systems have been devised. The use of thermo- couple psychrometers for measuring water potential in the field requires elaborate switching and data acquisition systems (37, 80). Gamma-ray transmission is less desirable because readings are of soil water content rather than of water potential, and sophisticated instrumentation is re- quired (93). Tensiometers measure matric potential and can be connected to a pressure transducer and recorder for continuous measurements (28). Although this equipment is inexpensive, tensiometers are capable only of measurements from 0 to -0.80 bar. Gypsum soil moisture blocks are the most inexpensive and reliable means of measuring a wide range of soil matric potentials. A block's im- pedance changes with the matric potential in the soil from -0.3 to -15 bars (9). The blocks are commercially available in many shapes (15, 87, 119) and can be easily made (81). Cylindrical blocks have been shown to have the least amount of variability at different suctions (15) and be- tween wetting and drying cycles (119). The impedance, which is mainly resistance and some reactance effects, is measured in ohms. Most of the instruments which measure resistance of the moisture blocks use an a.c. wheatstone bridge circuit With capacitors incorporated in the arms, to eliminate reactance so that a null point resistance reading can be made. These instruments are not suitable for continuous field readings because they require manual balancing. James Maine, of the Michigan State University Biochemistry Depart- ment, and I designed and built an inexpensive moisture block meter using an IC amplifier and mA meter which measures the voltage drop from an oscillator caused by the moisture block's impedance. The meter is calibrated to read in ohms. This circuit design eliminates the need for manual balancing and incorporates zener diodes to insure stable readings. Interfacing a two-channel analog recorder and switching system with the meter enables sequential automatic measurements of 12 blocks and con- tinuous recording of one block. Description of recording soil moisture meter: In the laboratory a :12 V d.c. 120 mA highly regulated power supply was used (Figure 1). In the field the power was supplied by two 12 V d.c. storage batteries in series which ran the equipment for over a month before needing to be recharged. Within the moisture meter box 10 V/1 w zener diodes were used to reduce the voltage to :10 V d.c., thereby giving only a 1.5% error in the total scale reading when the power supply voltage fluctuated as much as :2 V. The moisture blocks, 81 to 812, and 813 which were buried in the soil, were connected between the meter's oscillator and amplifier. Channel 1, C1 was connected to a 24 V step relay R1 allowing for as many as 12 blocks, Bl to 812 to be read and channel 2, C2 was hard wired to a single block, 813. The recorder's transport cam switch which was rewired to conform to the diagram was connected to a 12 V d.c. trigger relay R2. The amplifier outputs from C1 and C2 were connected to recorder pins 3 and 5, respectively. By making the connections as described, a continuous recording of C2 and a dashed line of C1 was obtained. After each dash, the next position on the step relay was read. A second relay box could be added to channel 2, thereby increasing the total number of blocks to 24. POWER SUPPLIES RECORDER RELAY BOX LABORATORY NO cm - - - ° ‘2' N I "'4‘” MN 0 I d 0 4 3 6 :5 7 8 r5 .... <7 (<7 I . l — -IE 81 «:12 FIELD ' 010v ‘ ov Hav -Iav Cl 7: N L - 1E unenv sunny "" -10v 7 o 0 O O OIZV AA "A”— .101! k c: 220 1r N ll: 1e are A -12v -IOV MOISTURE METER BOX Figure 1. A block diagram of the recording soil moisture meter. The recorder was an inkless Minigraph 12 V d.c., 0-1 mA d.c., analog recorder with a time-share two input feature (Esterline Angus In- strument Corporation, Indianapolis, Indiana 46224 U.S.A.). The recorder is available with several different gear ratios allowing the chart paper to be used at a rate of 0.16 cm to 15 cm per hr. Since the relay system is activated by the recorder, the frequency of the switching depends on the gear-train chosen, ranging from 6 min to 15 hr. Description of the moisture meter circuit: With use of an oscillosc0pe, VR1 and VR2 variable resistors in the oscillator circuit (Figure 2) can be adjusted to give a smooth sine wave over a given peak-to-peak voltage value with oscillations of 1 kHz. The peak-to-peak voltage determines the maximum resistance range that can possibly be measured on the 0-1 mA d.c. meter in the recorder. Appro- priate voltage values are indicated below for three resistance ranges. Voltage, Resistance Values (ohms) peak-to-peak range maximum resolution .25 0-47 k 100-10 k .25 0-1 k 33-1 k .65 0-330 k 1.5-33 k For each voltage chosen, many different resistance ranges can be read on the 0-1 mA meter. The resistance readings are non-linear; therefore, each range has an area of maximum resolution. The maximum resistance reading, which is zero on the meter, is determined by adjustment of VR3 in the amplifier circuit. Variable resistor VR4 is used to adjust the OSCILLATOR AMPLIFIER +10V -10V Figure 2. Circuit diagram of a single channel used in the recording soil moisture meter. (B) soil moisture block; (C1)(C2)(C3) 0.1 pF; (C4) 0.47 uF; (C5) 50 uF; (D1)(D2) IN58A; (D3) IN34A; (IC1)(IC2) 741 op. amp.; (M) 0-1 mA d.c. in recorder; (R1) 10 k; (R2)(R3)(R4) 1 k; (R5) 100 k; (VRl) 50 k; (VR2) 10 k; (VR3)(VR4) 50 k. 10 minimum resistance value to read as 1 on the meter. Both variable resis- tors were 10-turn to allow for fine tuning. This meter is designed as a resistance meter; however a gypsum soil moisture block does exhibit a small capacitance effect, therefore the meter responds to the block's impedance. Impedance readings of moisture blocks from the meter were only 20% higher than those readings from a Beckman_Model RC-lZClP soil moisture conductivity bridge (Beckman Instruments, Inc., Cedar Grove, New Jersey 07009) which measures pure resistance. However, whether measuring resis- tance (9, 59), capacitance (3) or impedance, a block's values change with the matric potential and therefore should be calibrated against a soil at known matric potential values. Performance: The recording soil moisture meter, and an optional temperature recorder were easily mounted in a styrofoam ice chest with inside dimen- sions of 25 cm wide, 37 cm long and 30 cm deep (Figure 3). The relay box was stored under the mounting board. The equipment was used continuously over the summer of 1978 to measure impedance of cylindrical soil moistureblocks with 15 foot leads obtained from Soilmoisture Equipment Corporation, Santa Barbara, Calif. 93105 U.S.A. The blocks' matric potential values were determined by em- bedding the blocks in field soil in a ceramic pressure plate appparatus. Over three, two-week field trials, with meter range 0-47k ohms, the readings of a fixed 100 ohm resistor did not change more than :1% of full scale (i 25 ohm) even though the temperature ranged from 12 to 34 C. The amount of drifting within each range decreases with increasing 11 Figure 3. Mounting of recording soil moisture meter and temperature recorder in styrofoam ice chest. 12 resistance values. A 100 ohm resistor calibrated at 24 C gave a 2% lower full-scale reading when subjected to 1 C and a 2% higher reading when placed at 45 C. Since the drifting is a given percent of full-scale, the accuracy of the reading depends on the range of resistance one cali- brates the 0-1 mA d.c. meter to read. The low current drain of 28 and 60 mA from the meter and recorder, respectively, allows the entire system to be operated on lead-acid storage batteries for several weeks. The two-channel feature gives the instrument versatility by allowing two different resistance ranges to be read simultaneously. The simplicity of the circuitry permits the meter to be economically constructed and maintained. PART II A LABORATORY METHOD FOR SCREENING POTENTIAL SOIL-BORNE PATHOGENS OF SOYBEAN INTRODUCTION There are many methods of screening soil-borne organisms for their pathogenic capabilities, each with advantages and disadvantages which have to be weighed before making a choice. The most standard method involves adding inoculum to a sterilized planting medium such as Vsoil or sand (41), but this method often requires a large amount of inocu- lum to produce disease. Schmitthenner (109) devised a pathogenicity test which requires less time and inoculum. The method consists of placing the inoculum as an agar layer in a pot containing soil, then covering the layer with additional soil and planting the seeds. The presence of the soil barrier between the agar layer and seed reduced pre-emergence damping-off. This method was used successfully in testing pathogenic isolates of Aphanomyces, Phytophthora, Pythium, Rhizoctonia and Fusarium against alfalfa and soybean hosts. Johnson et al. (52) modified this approach by placing the inoculum as a small agar disk next to the hypo- cotyl of cotton plants. The root-dip method, which consists of dipping the roots of the host into an inoculum suspension then transplanting into a suitable grow- ing medium, is very efficient in conserving inoculum (49, 95). The root-dip method is not always desirable because some plants, by the time they develop a substantial root system, may have become resistant to the pathogen (18, 71). 14 15 The most space-efficient methods use tubes or plates as containers. Kilpatrick (57) used test tubes (18 X 150 mm) as containers and damp fil- ter paper to support the inoculum disk and seedlings. Maduewesi and Lockwood (76) used test tubes which contained infested soil to evaluate isolates of ThieZaviopsis basicoZa. The easiest method consists of placing the seeds or seedlings directly on agar disks containing the test fungus. However, as a method becomes more space-efficient and simpler, it also becomes further removed from the field condition. Kerr's method (55) of using virgin soil infested with a culture of the test fungus in sterilized soil is about the closest one can get to the natural field condition; however, such a method is not convenient for testing many isolates. This section deals with a tube container method of screening a large number of soil-borne pathogens of soybean. MATERIALS AND METHODS Pythium ultimum Trow., Fusarium roseum (Lk.) emend Snyd. and Hans., F. oxysporum (Schl.) emend Snyd. and Hans., F. soZani (Mart.) Appel. and Wr. emend Snyd. and Hans., Rhizoctonia sp., and Thielaviopsis basicola Berk. and Br. were isolated from lesions on soybean plants. Fusarium oxysporum isolate 2, F. solani isolate 2, F. tricinctum (Cda.) emend Snyd. and Hans., F moniZifbrme 'subglutinans' Wollenw. and Reink. were isolated as saprophytes from dead tissue. Phytophthora megasperma var. sojae A. A. Hildebrand isolates 1 and 3 were obtained from Dr. A. F. Schmitthenner (Department of Plant Pathology, Ohio State University, 0. A. R. D. C., Wooster, Ohio 44691). The Fusarium species were identi- fied by Dr. Paul E. Nelson (Department of Plant Pathology, Pennsylvania State University, University Park, PA 16802). P. uZtimum, Fusarium species, and Rhizoctonia 5p. were grown on potato-dextrose agar (PDA). Phytophthora megasperma var. sojae was main- tained on 'Difco' lima bean agar. Thielaviopsis basicola was grown on potato-yeast extract agar (extract of 200 g potato, 5 g yeast extract, 30 g dextrose and 20 g agar per liter). The soybean (Glycine max (L.) Merrill cv. Hark and Harosoy 63) were surface-disinfected with propylene oxide (104) overnight and allowed to dry for at least one day. Harosoy 63 soybean seeds were used when Phytophthora isolates were tested. The seeds were planted in 16 17 polystyrene fir tree seedling tubes 12 cm X 2.5 cm diameter (Ray Leach's "Cone-tainer" Nursery, Canby, Oregon 97013). The tubes' side drainage holes were covered with Time adhesive tape and the bottom holes were capped with a piece of nylon mesh (250 pm) secured by an inner and outer ring of Tygon tubing (Figure 4). Two seeds were placed on top of a 17 mm diameter agar disk of the test fungus which was one to two weeks old. The agar disk rested on a 4 cm layer of a given medium [sand, a mixture of vermiculite and perlite (7:3 v/v), or vermiculite] and the agar disk and seeds were covered with a layer of medium (5 mm sand covered with 10 mm perlite, 4 cm vermiculite, or 4 cm of the vermiculite-perlite mix- ture). The tubes were watered once daily until they drained freely and were incubated at 25 C for 11 days under continuous fluorescent light (10,000 lux). Four tubes were used to evaluate each isolate. Disease severity was assessed visually using a separate rating system for those isolates causing seedling damping-off and those pro- ducing root rot. The seedling disease severity scale consisted of: (0) for no lesions, (1) superficial lesions, (2) severe necrotic lesions ex- tending to the pith of the primary root, (3) post-emergence damped-off, (4) preeemergence damped-off. The root rot rating was used to evaluate those isolates which did not cause damping-off and is as follows: (0) no lesions, (1) slight lesions, (2) one severe lesion or 5-10% of the root discolored, (3) two severe lesions or more than 10-25% of the root dis- colored, (4) 25-60% of the root discolored, (5) greater than 60% root discoloration. The disease index was the mean rating of four tubes. 18 * BEAN ED AGAR DISK ’QQ‘L SOY SE I I DRAIAISIEKGEOVIESLE \\ 7! 33(5):. Rug” : INNER R1N§__ Figure 4. Cross-section of a polystyrene tube used for assessing pathogenicity of fungal isolates to soybean. Various media were placed above and below the seed and inoculum agar disk. RESULTS Inspite of the large apparent differences between some of the planting media disease indices, few were statistically significant be- cause of the large amount of variability among replications. Thielaviop- sis basicola root rot was slightly favored when vermiculite was used for both top and bottom layers (Table 1). When the different planting media were compared using seedling rotting isolates, again, there were few statistically significant dif- ferences in the disease indices except in the case of Fusarium solani isolate 2 (Table 2). Only superficial lesions were caused by F. solani isolate 2 when a mixture of perlite and vermiculite was used for both layers as compared with more severe symptoms with the other media. Most of the Harosoy 63 soybean seedlings failed to emerge in any of the media when inoculated with race 3 of Phytophthora megasperma var. sojae to which this variety is susceptible. Harosoy 63 is resistant to race 1 and emerged normally when inoculated with race 1. However, in the case of several other differential varieties tested, susceptible reactions were obtained when resistant reactions were expected. In another set of experiments, only the planting medium with sand as the bottom layer and vermiculite-perlite mixture as the top layer was used (Table 3). Whether these tubes were watered every day or every other day, or incubated for the first four days in the light or dark, 19 20 Table 1. Severity of soybean root rot caused by five fungal isolates used to infest five different planting media in plastic tubesa . Disease index for indicated planting mediumb Fungi V/V VP/VP S/S/P S/V S/VP Fusarium oxysporum #1 1.75 aC 1.25 a 2.75 a 2.88 a 2.13 a Fusarium oxysporum.#2 1.00 a 1.38 a 2.13 a 1.75 a 2.25 a Fusarium soZani #1 0.75 a 0.50 a 1.75 a 1.00 a 1.63 a Fusarium roseum 2.00 a 1.38 a 1.63 a 1.50 a 1.13 a ThieZaviopsis basicola 4.75 a 3.38 ab 2.75 ab 2.63 ab 2.38 b aData are means of 4 replicated tubes, each with 2 seedlings. Disease was evaluated on a scale of increasing severity from 0-5. bPlanting media: seeds were planted on the surface of the bottom layer and covered with one or more layers: vermiculite (V), vermiculite- perlite mixture 7:3 v/v (VP), fine sand (5), and perlite (P). CMeans for the same fungus followed by the same letter are not signifi- cantly different from each other (P_= 0.05) using Tukey's g_procedure on transformed data: J x + 0.5 . 21 Table 2. Severity of soybean seedling rot caused by six fungal isolates used to infest five different planting media in plastic tubesa Disease index for indicated planting mediumb Fungi V/V VP/VP S/S/P S/V S/VP Fusarium solani #2 1.63 abc 0.50 b 2.88 a 1.50 ab 2.25 a Fusarium tricinctum 4.00 a 4.00 a 3.55 a 3.35 a 3.38 a Fusarium monilifbrme 'subglutinans' 3.13 a 3.25 a 3.63 a 3.38 a 3.25 a Pythium ultimum 2.13 a 3.38 a 4.00 a 2.75 a 3.50 a Phytophthora megasperma var. sojae race I 0.00 a 0.25 a 1.00 a 0.00 a 0.75 a Phytophthora megasperma var. sojae race 3 4.00 a 3.75 a 2.25 a 2.00 a 2.00 a aData are means of 4 replicated tubes, each with 2 seedlings. Disease was evaluated on a scale of increasing severity from 0-4. bPlanting media: seeds were planted on the surface of the bottom layer and covered with one or more layers: vermiculite (V), vermiculite- perlite mixture 7:3 v/v (VP), fine sand (5), and perlite (P). cMeans for the same fungus followed by the same letter are not signifi: cantly different from each other (E_= 0.05) using Tukey's w_procedure on transformed data: 7 x + 0.5 . 22 Table 3. Severity of root rot of soybeans planted in plastic tubes caused by five fungal isolates under four lighting and water- ing schedulesa Disease index for indicated lighting and wateringscheduleb Fungi NL L W0 0 Fusarium oxysporum #1 2.06 ac 1.84 a 1.94 a 2.25 a Fusarium oxysporum #2 2.18 a 2.48 a 2.54 a 2.45 a Fusarium solani #1 1.31 a 1.29 a 0.68 a 1.49 a Fusarium roseum 1.38 a 0.97 b 2.35 a 2.32 a Thielaviopsis basicola 2.19 b 2.09 b 1.88 c 2.89 a aData are means of 5 replicated tubes, each with 2 seedlings. Disease was evaluated on a scale of increased severity from 0-5. bSchedule: watered every day and incubated under light (WL), watered every other day under light (L), watered every day and incubated in darkness 4 days (WD), watered every other day and incubated in darkness 4 days 0). CMeans for the same fungus followed by the same letter are not signifi- cantly different from each other (P_= 0.05) using Tukey's g procedure on transformed data: J x + 0.5 23 made little difference in the amount of seedling and root rot. Root rot caused by T. basicoZa was favored slightly by placing the plants in the dark for the first four days and watering every other day. F. roseum caused the least amount of root rot when the plants were placed under the light and watered every other day. Seedling rot by F monilifbrme 'sub- glutinans' was most severe under continuous light with daily watering (Table 4). 24 Table 4. Severity of seedling rot of soybeans planted in plastic tubes caused by seven fungal isolates under four lighting and water- ing schedulesa Disease index for indicated lighting and watering scheduleb Fungi WL L WD 0 Fusarium soZani #2 1.09 ac 0.77 a 0.51 a 0.61 Fusarium tricinctum 3.99 a 3.99 a 3.79 a 3.79 Fusarium monilifbrme ‘subglutinans' 3.89 a 3.48 b 2.87 c 3.48 Pythium uZtimum 3.58 a 3.58 a 3.55 a 3.99 Phytophthora megasperma var. sojae race 1 1.86 a 1.09 a 1.64 a 1.47 Phytophthora megasperma var. sojae race 3 4.00 a 4.00 a 4.00 a 4.00 Rhizoctonia spL 1.92 a 2.81 a 2.38 a 3.13 3Data are means of 5 replicated tubes, each with 2 seedlings. Disease was evaluated on a scale of increased severity from 0-5. b Schedule: watered every day and incubated under light (WL), watered every other day under light (L), watered every day and incubated in darkness 4 days (WD), watered every other day and incubated in darkness 4 days (0). cMeans for the same fungus followed by the same letter are not signifi- cantly different from each other (P_= 0.05) using Tukey's w_procedure on transformed data: J x + 0.5 . DISCUSSION The best planting medium which gave good lesion devel0pment with each fungus was 4 cm of sand as the bottom layer and 4 cm of vermiculite- perlite mixture covering the seeds. Placing the plants under continuous light and watering every day is recommended because it seems to favor the growth of the control seedlings more than the other lighting and watering schedules. This method was primarily developed so a large num- ber of isolates could be screened for pathogenicity. Sixteen inoculum agar disks can be cut from one 9 cm diameter petri dish. The system probably overestimates the virulence of the pathogen because of the' placement of the seeds directly on the agar disk and the absence of in- hibiting organisms. The placement of the seeds directly on the agar disks may have influenced disease development to such as extent that planting medium, watering or light made little difference. The method was developed primarily for testing Fusarium Species, which have low pathogenic capabilities on soybean (126). 25 PART III COLONIZATION OF PLANT TISSUE AND SOYBEAN SEEDS BY FUSARIUM SPECIES IN NATURAL AND ARTIFICIALLY INFESTED SOIL INTRODUCTION Sinclair and Dhingra (111) in their "Annotated Bibliography of Soybean Diseases, 1882-1974" listed 72 papers which cited a total of 19 species of Fusarium as being pathogenic to or frequently isolated from soybean. Fusarium oxysporum is usually cited as the main pathogenic species. It causes Fusarium blight and Fusarium root rot. The first report of a Fusarium species causing a disease of soybean in the United States was made by Cromwell (27), who reported a blight disease of soy- bean caused by F. tracheiphilum (F. oxysporum). This Fusarium also caused a wilt of cowpea. 0n soybean, the first symptom was discolora- tion of the root xylem tissue which occurred 8 weeks after planting. Later, general yellowing and dwarfing of the plant occurred with vascu- lar discoloration extending into the stem. The blight or wilt disease usually occurred in the southern states (123) and was favored by high temperature (28 C). Fusarium wilt of soybean can be caused by more than one race of F. oxysporum (4, 5, 6). F. oxysporum isolates which cause root rot symptoms may also cause poor germination and stunted plants (31). Rarely are plants be- yond the seedling stage killed. In Minnesota, French and Kennedy (39), using the inoculum agar layer technique to test the pathogenicity of 28 Fusarium isolates, found 14 of 18 F. oxysporum isolates were pathogenic whereas 8 F. soZani and 2 F. moniZifbrme isolates were nonpathogenic. 27 28 Warren and Kommedahl (126) obtained isolates from roots, plant residues, rhizosphere soil and soil from monoculture soybean plots and found three times more F. oxysporum than F. soZani which was the next most prevalent species. Root rot caused by F. oxysporum is generally favored by low soil temperature (14 to 23 C) plus high soil moisture (saturated) (31, 39). However, high temperature, 27 C, and low moisture, 40% field capa- city, may also favor disease devel0pment (31). The ecology and patho~ genicity of Fusarium species from soybean have not been thoroughly studied, even though they are a major cause of soybean root diseases which are responsible for a significant reduction in soybean production (112). Fusarium species are known to be able to grow and colonize steri- lized as well as nonsterilized material over a wide range of water poten- tials. Germination of Fusarium roseum f: sp. cerealis 'Culmorum' chlamydospores in soil was reduced to only 40-50% at potentials of ~50 to ~60 bars and did not cease until ~80 to ~85 bars (24). On agar, my- celial growth was reduced to one half at ~50 bars osmotic potential (25). The optimum water potential at which it will grow on osmotically-adjusted agar depends on the temperature: ~8 to ~14 bars at 20 C to 30 C, and ~28 bars at 35 C (22). Colonization of straw by F. roseum f: sp. cereaZis was maximal at ~50 to ~60 bars but also occurred at ~0.5 to -1 bar water potential (20). Growth on wheat straw ceased at ~70 to ~75 bars matric potential (25). Fusarium soZani ft pisi chlamydospores had maximal ger- mination (46%) in soil contiguous to germinating pea seeds at water po.- tentials between ~0.3 and ~15 bars (23). Kouyeas (66) reported that sterilized wheat straw pieces and tomato root fragments were colonized by Fusarium Species when placed in field soil from ~0.45 to greater than 29 ~°0 bars water potential. F. oxysporum colonized sterilized human hair at 100% relative humidity (RH) as well as at 90% RH (16). A soil-borne pathogen such as Fusarium would have an advantage under dry soil condi- tions because of the reduced growth of other soil microbiota which would normally compete with Fusarium, and the host might also be stressed un- der dry conditions which would favor Fusarium. The survival structure of Fusarium is usually considered to be the chlamydospore which usually survives in buried plant tissue or other organic matter (17, 84, 125). F. oxysporum f2 cubense which causes wilt of banana is usually only found in cortical tissues of decayed host roots and not free in the soil (122). It was recovered from soil after a longer period of time when colonized glass cloth strips were placed in the soil than when a cornmeal-in-sand culture was used as inoculum (86). Fusarium propagules are generally found to be unevenly distributed in the surface soil (1, 122). If the soil is plowed the propagules may be more evenly distributed in the plow layer (10, 83). F. soZani chlamydospores require a carbon and nitrogen source for germination (26) and survive longer in dry soil (air dry to 42% water holding capacity) than in satu- rated soil (17, 23). Population densities of F. oxysporum f: cubense, f2 nicotianae, f; niveum, F. moniZifbrme and F. graminearum were also noted to decrease in saturated soil (116). It has been extensively reported that various Species of Fusarium will colonize dead plant tissue in soil. However, there have been few research papers dealing with the influence of such colonization on the Fusarium inoculum density of the soil. 30 In this section, I will report on the saprophytic colonization of plant tissues as a means of survival of pathogenic Fusarium species. Since Fusarium species will colonize dead tissue and are often seed-borne (54, 58, 85, 101), I also tested the influence of Fusarium colonization of the seed on emergence. MATERIALS AND METHODS Estimation of Fusarium population: Komada's~Fusarium~selective medium was used for enumerating Fusarium population density in plant tissue and soil (64, 65). Soil dilutions were made by adding approximately 10 grams of soil (dry weight basis) to a flask containing 90 ml of 0.075% water agar. The flask was placed on a shaker (143 cycles/min) for 15 to 30 minutes. The soil sus~ pension was diluted as needed (10'2~10-“) by transferring 1 ml of suspen- sion to 9 ml of water agar. One ml of the appropriately diluted soil SUSpension was spread over the surface of each selective medium plate. Four plates were used for each dilution. The numbers of propagules of Fusarium per cm2 of surface area of plant tissue also was determined. Tissue pieces with adhering soil were placed on a shaker for 30 min. The tissue pieces were then recovered by filtering through one layer of cheese cloth and the suspension plated. The weight of adhering soil was determined by centrifuging the solution, discarding the supernatant; and‘ drying the sample at 95 C. The clean tissue pieces were measured (length and width), cut up and minced for 2 min in a Waring blendor at low speed with 90 ml 0.075% water agar. One ml of this suspension was diluted as necessary and plated on the selective medium. After the water was absorbed by the agar, the plates were incubated in plastic bags on a laboratory bench at 23 1 2 C under ambient light (5 to 8 lux during 31 32 the day). After 5 to 7 days colonies were tentatively identified on the plates based on pigmentation, colony morphology, size of conidio- phores and general conidial shape, using the classification of Toussoun and Nelson (121). Specific isolates were identified by either Paul E. Nelson (Department of Plant Pathology, Pennsylvania State University, University Park, PA 16802) or Lester W. Burgess (Department of Plant Pathology, University of Sydney, Sydney, N.S.W. 2006, Australia). Isolates were maintained on PDA at 5 C in screw-capped vials and on sterile (propylene-oxide treated) soybean pod tissue cultures main- tained in a desiccator at 23 i 2 C. Propylene oxide treatment of soil, seeds and dry plant tissue: The soil (1 liter) was disinfested by moistening it with water (~0.4 t 0.2 bar) in a 1.9 liter glass jar, adding 2 ml propylene oxide and sealing the jar for 2 days. The jar was opened, aired under a hood overnight and then dried in a forced air oven at 35 C. Disinfected soybean seeds of the variety Hark were used throughout this study. The seeds (IOU-200) to be disinfected were placed in a 500~ ml jar. While under a fume hood, a small test tube containing 0.5 ml of pr0pylene oxide was placed inside the jar, which was tightly sealed with a rubber stopper for 9 to 16 hr (104). The stopper was removed and a piece of loose-fitting aluminum foil was placed over the jar opening for a day. After treatment, only 3% of the seeds yielded Fusarium Species when plated on Komada's selective medium. Mature soybean stem pieces (3.0 i 0.5 cm X 0.4 i 0.2 cm) were placed in a 1.9 liter glass jar on a piece of moist paper toweling, two ml of propylene oxide were added and the jar was sealed for 24 hr (43). 33 Stem pieces were removed, aired for 24 hr in a forced-air oven at 30 C, and treatment was repeated. After processing, some of the stem pieces were removed, split open and plated on Komada's medium and PDA to check for contamination. No fungal contaminants were found following treat- ment. Pathggenicity test for Fusarium isolates: Isolates were evaluated in 2.5 X 12 cm polystyrene tubes with drainage holes. Soybean seeds were disinfected and 2 seeds were placed on a 17 mm diameter disk of the test isolate which in turn rested on a 4 cm deep layer of fine washed, white silica sand (Figure 4). The bottom drainage hole was covered with nylon netting to retain the sand and the side holes were taped over with Time adhesive tape. Seeds were covered with a 4 cm layer of a mixture of venniculite and perlite (7:3, v/v). The tubes were watered daily and incubated at 25 C for 11 days under continuous cool white fluorescent light (10,000 lux) or in the greenhouse. Virulence was rated using the seedling disease index as outlined in Part I. Soil characteristics and matric potential determinations: Unless otherwise indicated, all soil used in the laboratory ex~ periments was from the field in which field experiments were done. It was a sandy loam soil with the following characteristics: sand:silt:clay = 80.9:4.1:15.0, pH 5.5, 1.78% organic matter, and exchangeable bases (K:Ca:Mg) in the ration 3.9:81.1:15.0. Soil moisture-matric potential curve (Figure 5) was determined by using a 15 bar ceramic plate extractor (Soilmoisture C0rp., Santa Barbara, Calif. 93105) with 1 and 15 bar 34 I-BAnsl MATRIC POTEN TIAL oOOl l l l 2 4 a 8 101214 1618202224 26 PERCENT son. MOISTURE (oar WEIGHT BASIS) Figure 5. Soil moisture-matric potential curve of a sandy-loam soil obtained by desorption with a pressure plate apparatus. 35 plates (96). The soil was dry-sieved (4 mmz) and placed in retainer rings (5.5 cm diameter X 1 cm height) each resting on a 10 cm2 piece of nylon mesh (250 pm) on the ceramic plate. The plate with soil was saturated overnight before applying the pressure. After the water flow from the extractor stopped, the soil was removed from the mesh and placed in a drying can for 24 hr at 95 C. Once the soil moisture- potential curve was determined, a given soil matric potential was estab- lished by atomizing water into the dry soil in a plastic bag with fre- quent Shaking until a predetermined percent soil moisture level was reached. Saprophytic survival in nonsterile tissue in the field: Saprophytic survival of Fusarium in infected host tissue was de~ termined by burying 6 mature soybean root tissue pieces (roughly 2.5 cm X 0.5 cm) with Fusarium root rot symptoms in 250 ml of natural Fusarium- infested soil in nylon mesh bags (250 pm meshes). The bottom of the mesh bags containing the soil and tissue pieces were placed on the soil surface or at depths of 7.6 and 15.2 cm. Three small plots within the field were used as blocks. At this same location separate mesh bags of soil containing either mature corn stalk, wheat stems, soybean pods or soybean stem tissue without Fusarium rot Symptoms were buried 15.2 cm deep. Influence of soybean residue on the bulk soil population: Two weeks after a soybean crop harvest, small portions of the soil were sieved (4 mm meshes) to remove plant residues and buried in nylon mesh bags (800 g) 15.2 cm deep in the field. This constituted the 36 low residue treatment. The residues which were retained on the sieve were added to the unsieved field soil and buried. This constituted the high residue treatment. Unsieved field soil was used as the medium level residue soil. After 7 and 9 months, the residue pieces were removed by sieving (4 mm meshes) and Fusarium populations of the soil were deter- mined. Saprgphytic colonization of disinfected tissue: Two disinfected soybean stem pieces (2.5 cm X 0.5 cm) were placed in 200 g of soil at a given matric potential in plastic cups (530 ml) which were placed in plastic bags in an incubator at 20 C. The control soil was maintained at ~90 bars. The stem pieces were removed at given time intervals. Fusarium population associated with the tissue and the adhering soil (ca. 10 9 soil) were then detennined as outlined before. Each stem constituted a subsample, and the experiment was repeated twice for a total of 3 blocks. To determine the sphere of influence the tissue has on the Fusarium population, disinfected soybean stem pieces were buried in a pan (14 cm X 19 cm) of soil (~1.8 bar) at 20 C. A piece of styrofoam was placed in the bottom of the pan and thin insect pins were used to hold the stem pieces and to mark 3 mm distances from the stem surface. Just enough soil was added to the pan to just cover the horizontally lying stem pieces. The pan was placed in a plastic bag to reduce evap- oration. After 16 days the soil was watered with an atomizer until the soil could be sliced and then sections were removed with a razor blade at the marked distances from the stem pieces for population determina~ tions. 37 Preemergence rotting of soybean seeds by Fusarium: Twenty disinfected seeds were planted 2.5 cm deep in soil at specified matric potentials in a rectangular plastic tray (19.6 X 9.7 X 5.5 cm), double-bagged in plastic and incubated at 24 C. The experiments were replicated three times unless indicated otherwise. Soil matric potential was increased to -0.24 bar after a given time period by re- moving the seeds, placing the soil in a plastic bag and mixing while adding water with an atomizer. The soil was added back to the trays and the seeds planted. To effectively control either Pythiaceous fungi or Fusaria, Lesan (Dexon) (sodium [4-(dimethylamino)phenyl] diazene sulfonate) (48, 55, 70, 118) or benomyl (methyl 1-[butylcarbamoyl]~2~benzimidazolecarbonate) (34, 48), respectively, were added to the soil. The chemicals were sus- pended in water and atomized into the soil which was allowed to dry on paper toweling. Final concentrations were 30 mg Lesan and/or 300 mg benomyl, a.i.,lkg soil. Soil with a single Fusarium Sp. was established by adding 5 ml of water to 1-2 week-old Fusarium cultures on PDA and sprinkling the coni- dial suspension onto dry, disinfested soil until the moisture was 0.5 i 0.2 bar matric potential. This soil was stored at 23 2 2 C in a plastic bag for at least one week before it was allowed to air dry. The Fusarium population was determined after drying. Soil was then diluted with disinfested soil so that a population of 3000 propagules per g (PDQ) was established. RESULTS Saprophytic survival in soil and nonsterile tissue: The number of propagules of Fusarium in the surface soil (0-3 cm) did not change Significantly over a 9-month sampling period (Table 5). However, the number of propagules in Fusariam-infected mature soybean root pieces which were placed on the soil surface did change signifi- cantly (P_= 0.10) over the sampling period, the highest level occurring in October. There were no differences in the proportion of different Fusarium species recovered over the same period. F. oxysporum accounted for 80.8% of the Fusarium colonies; F. solani, 15.5%; and the other Fusarium species, 3.7%. When the root tissue was plated, 92% of the colonies were F. oxysporum; F. soZani and other fungi constituted 6.2 and 1.8%, respectively. F. oxysporum accounted for 95% of the Fusarium isolates which were recovered from lesions from 120 young soybean plants, which were surface sterilized for 1 min in 0.5% NaOCl. No differences were detected in the pathogenicity to soybean seedlings of 40 isolates of F. oxysporum obtained from buried root tissue as compared with 40 isolates from soil (disease indices of 2.0 and 2.4, respectively). There was no significant difference (P_= 0.05) in the Fusarium population density at 7 and 9 months at three depths; therefore, the data were combined for analysis. The number of propagules associated 38 39 Table 5. Population density of Fusarium in a sandy-loam soil and in mature root tissue collected at four different times. Number of propagules/g soil Substrate or cm2 root surface (X103)a September October April June 5011 4.8 ac 6.0 a 7.6 a 3.9 a Rootb 5.2 a 79.3 b 10.1 a 9.4 a aMeans of three blocks. bMature soybean root pieces with Fusarium root rot symptoms were placed on the soil surface in nylon mesh bags in September. CMeans for the same substrate followed by the same letter are not signi~ ficantly different from each other using Tukey's w procedure (P.= 0.10) on loglox transformed data. 40 with the soil or root pieces did not change with soil depth at 0-3 cm, 7.6 cm and 15.2 cm (Table 6). However, the number of propagules in the soil adhering to the tissue was higher at the soil surface than at depths of 7.6 or 15.2 cm [1.3 X 105, 2.4 X 10“ and 3.2 X 10“ propagules per g (ppg), respectively]. The p0pulation in the soil which adhered to the pieces was significantly (E_= 0.05) higher than that in plain soil at all depths (statistical analysis not shown in Table 6). The population density of Fusarium in soil which adhered to tis- sue pieces after they were buried for 9 months was 6-27 times higher than that of soil which was not associated with the tissue (Table 7). From each tissue type, 20 F. oxysporum isolates were obtained and their pathogenicities were compared with isolates from field soil. No significant (P_= 0.05) differences were detected among the seedling dis- ease indices (soybean pods, 2.0; soybean stems, 1.5; corn, 2.1; wheat, 1.9; and field soil, 1.7). Removing post-harvest soybean residue from soil or doubling the amount of residue did not Significantly (P_= 0.05) change the Fusarium population of the soil after 7 or 9 months. Low residue soil had a Fusarium population density of 5.6 X 103 ppg; unaltered field soil, 6.2 X 103 ppg; and high residue soil, 4.4 X 103 ppg. Saprophytic colonization of disinfected tissue: To determine if Fusarium species would colonize tissue pieces which have not been previously colonized by Fusarium or other fungi, disinfected soybean stem pieces were added to field soil and incubated in the laboratory for 8, 16 and 32 days at ~0.016,~1.8 and ~90 bars (extrapolated) matric potentials (Table 8). The only treatment which did 41 Table 6. P0pulation density of Fusarium in a sandy-loam soil and in mature root tissue pieces buried at three different soil depths for 9 months. Number of propagules/g soil or cm2 root surface (X103)a Substrate 0-3 cm 7.6 cm 15.2 cm Soil 5.8 ac 3.3 a 5.1 a Rootb 9.7 a 8.4 a 5.5 a Soil adhering to root 132.3 b 23.9 a 31.6 a aMeans of three blocks combined over two sampling periods (7 and 9 months). bMature soybean root pieces with Fusarium root rot symptoms were buried in soil in nylon mesh bags in September. Adhering soil was removed from the root pieces by shaking in water agar. cMeans for the same substrate followed by the same letter are not signi- ficantly different from each other using Tukey's w_procedure (E_= 0.05) on loglox transformed data. 42 Table 7. Population density of Fusarium in a sandy-loam field soil and soil which adhered to plant tissue pieces after they were buried for 9 months in the field. Soil samplesb Number of propagules X103/g soila Field soil (control) 5.1 a Corn stalks 127.0 b Soybean stems 50.3 b Soybean pods 138.7 b Wheat straws 58.2 b Soybean roots with Fusarium root rot 31.6 b aMeans of three blocks combined over two sampling periods (7 and 9 months). Tissue pieces were buried in September in nylon mesh bags at a depth of 15.2 cm. CMeans followed by the same letter are not significantly different from each other using Tukey's w_procedure (E_= 0.05) on loglox transformed data. 43 Table 8. Population density of Fusariwm in soil which adhered to disin- fected soybean stem pieces buried in the soil at 3 matric poten- tials and in soil not associated with stem pieces at ~90 bars (control) sampled at three time periods. Number of propagules X103 Matric potential ~bars Days 8 16 5 32 ~0.016 25.1 bb 38.0 bc 30.9 b ~1.8 36.3 bc 104.7 cd 269.2 d ~90 19.1 ab 58.9 bc 35.5 bc ~90 control 4.2 a 8.5 a 10.8 a aMeans of three blocks. bMeans followed by the same letter are not significantly different from each other using Tukey's g_procedure on loglox transformed data. 44 not have a Significantly (P_= 0.05) higher population density than the control soil (7.2 X 103 ppg) was the stem pieces buried for 8 days at ~90 bars matric potential (1.9 X 10“ ppg). Even though the adhering soil population at 8 days and ~90 bars was not Significantly higher, the number of propagules per cm2 of tissue area which was 0 at the start had increased to 1.6 X 101+ indicating that Fusarium species had colonized the disinfected tissue. The population density of Fusarium in the soil adhering to the stem tissue was significantly (E_= 0.05) higher at ~1.8 bars (1.4 X 105 ppg) than at ~90 bars (3.8 X 10“ ppg) or ~0.016 bar (3.1 X 101+ ppg). After 16 and 32 days the population was significantly (P_= 0.05) higher (6.7 X 10“ and 1.1 X 105 ppg, respectively) than after 8 days (2.7 X 10“ ppg). The interaction between matric potential levels and incubation time was not Significant (P_= 0.05). F. oxysporum accounted for 88% of the Fusarium isolates and F. solani, 8%. There were no significant differences between the pathogenicity of F. oxysporum isolates obtained from field soil as compared to those from soil ad- hering to soybean stem pieces (disease indices of 2.3 and 2.6, reSpec- tively). Since F. oxysporum was the most prevalent Fusarium species iso- lated from soil and buried plant tissue, its soil population was moni- tored at given distances from disinfected stem pieces after 16 days. The F. oxysporum population density 0-3 mm from the stem piece (1.0 X 10“ ppg) was significantly (P_= 0.05) higher than that in control soil (4.1 X 103 ppg). At 3-6 mm and 6~9 mm distances, the densities were 4.4 X 103 and 2.6 X 103 ppg, respectively, which were not Significantly different from the control. 45 Preemergence rotting_of soybean seeds by Fusarium: There was a significant reduction in the emergence of seedlings when the seeds were placed in soil at ~15 bars for 4 days before watering to a level sufficient for germination (~0.24 bar) (Figure 6). The ex- periment was replicated over time with the same soil sample. F. oxysporum isolates accounted for 8 % of the Fusarium species isolated from seed buried 6 days. The same sample of soil was used in another experiment but the soil was treated with Lesan or Lesan and benomyl. Lesan was added as a soil treatment to avoid any preemergence damping-off caused by Pythium Species. Benomyl was added to remove the influence of Fusarium. The seeds were again left in soil at ~15 bars for 4 and 6 days before water- ing to ~0.24 bar. There was a Significant (P_= 0.05) reduction in ger~ mination at 4 days (32%) and 6 days (17%) in the Lesan soil as compared to Lesan and benomyl-treated soil after 4 days (80%) and 6 days (83%). In a similar experiment fresh soil was used for each replication, thereby eliminating any Fusarium population increase due to continuous plantings. After 6 days the seeds held at ~32 bars in Lesan-treated soil were the only ones that had reduced emergence (Table 9). The plants in soil treated with Lesan and benomyl consistently had better emergence than those in soil treated with Lesan alone. A sandy-loam soil collected from another farm which had a Fusarium population density of 2.1 X 103 ppg was treated with Lesan or Lesan and benomyl before adjusting moisture to ~32 bars and planting. Seeds were left in the soil for 8 days before watering to ~0.24 bar. Those planted in soil treated with benomyl and Lesan germinated signifi- cantly (P_= 0.05) better (95%) than those in soil treated with Lesan PERCENT EMERGENCE Figure 6. 46 -\!v' '1‘ 1' "-"' ".- ‘4- -"'."".'-,- 0 inf, .! -11.», '35:" .fl .1', ‘_:- .rf . 1 ‘L"1 .._ v «‘5 . u-‘ ~< 1,1. . .4... -. 31' §‘.. . 7.. .. '3‘ .‘ '~ \“ .1 Y '_.. DAYS AT '15 BARS Emergence of soybean seedlings from seeds sown in a sandy- loam soil held at ~15 bars for 0, 2, 4 and 6 days before watering to ~0.24 bar. Values are based on three consecutive plantings of 20 seeds per treatment in the same soil. Means followed by different letters are significantly different from each other using Tukeéfs w_procedure E_= 0.05 on transformed data: x + . 47 Table 9. Emergence of soybean seeds planted in a sandy-loam field soil treated with Lesan or with Lesan and benomyl and held for 6 days at 5 different matric potentials before watering to ~0.24 bar. a Matric potential Percent emergence ~bars Lesan Lesan + benomyl 2 93 ab 97 a 4 88 a 90 a 3 83 a 92 a 16 92 a 93 a 32 42 b 92 a aMean of three replications of 20 seeds each. bMeans followed by different letters are Significantly different from each other using Tukey's w_procedure (P.= 0.05) on transformed data: x + . 48 alone (7 %). Fusarium, AspergiZZus, Trichoderma, and PenciZZium Species were consistently isolated from rotted seeds washed and plated on water agar and potato-dextrose agar. Seven Fusarium isolates were tested for their ability to reduce soybean seed emergence when planted in soil at ~15 bars for 6 days. All the isolates had seedling disease indices of greater than 2.0. The per- cent emergence of those seeds planted in propylene oxide-treated soil at ~15 bars was 93%. Emergence in soil infested with one isolate of F. oxysporum and two isolates of F. solani was 85%, which did not differ Significantly from 93% (E_= 0.05). One isolate of each of the following fungi Significantly reduced germination to the indicated levels: F. soZani, 40%; F. tricinctum, 53%; F. monilifbrme 'subglutinans', 72%; F. oxysporum, 80%. DISCUSSION The soil population in our field remained relatively constant over the four sampling periods from September to June (Table 5). Nash and Snyder (83) reported similar results in California. They found that the population of F. soZani f1 phaseoli in soil did not differ from September to April in a field cropped to beans. Burke (10) also found relatively constant populations of F. soZani f: phaseoli in a bean field in Washington. Other researchers have reported that the population of Fusarium oxysporum will vary over the season with the lowest population occurring in the beginning of the season (78, 92, 94). F. oxysporum was the predominant Species isolated accounting for 81% of the isolates. F. oxyaporum was also found to be the predominant Species in a monocul- ture field of soybean in Minnesota (126). The number of propagules of Fusarium in infected mature root tis- sue pieces changed over a 9-month period at the 10% level of Significance. The highest level occurred in October which was 15 times higher than the starting September population. Indications are that the soybean root pieces were not completely colonized at maturity, and that the increased population may have been due primarily to conidia which would not sur~ vive as well as chlamydospores. F. oxysporum comprised 92% of the Fusarium isolates obtained from the root tissue which was Similar to the 84% reported by Ferrant and Carroll (35). 49 50 The Fusarium population density in soil and in Fusarium~infected root tissues did not vary with soil depth from 0 to 15 cm. The even dis- tribution of Fusarium propagules in the plow layer has also been re- ported for Fusarium solani f2 phaseoli (10, 83). There were signifi- cantly (P_= 0.05) more propagules in the soil which adhered to the tis- sue pieces than in the soil alone. The root tissue pieces placed on the soil had a higher population than those buried. The increased numbers of propagules may have been due to greater sporulation on the tissue pieces or improved survivability of the spores which may have resulted from better aeration or the availability of light on the soil surface. The presence of a higher population associated with tissue on the soil surface may be reSponsible for increased populations in no-till farming (35). Fusarium oxysporum colonized tissue which had not been previously colonized by Fusarium such as wheat straw, corn stalk, soybean pods and stems and caused a Significant increase in the number of propagules in_ the soil which adhered to the tissue. The population of pathogenic as well as non-pathogenic F. oxysporum isolates increased. The Sphere of increased population is less than 3 mm from the tissue surface and occurred within 8 days at ~0.016 to ~1.8 bars matric potential. When soybean harvest residue was added to soil and buried 15 cm, the bulk soil population (sieved soil) of Fusarium did not increase despite the fact that Fusarium proliferated on the tissue pieces. This is probably explained by: a) the lesser increase in population density occurring on buried residues as compared with those on the soil surface, b) the lack of physical mixing of residues with the soil during the time of the experiment, c) the small proportion of the soil volume occupied 51 by the soil adhering to the residue pieces. If the residue pieces had been incubated nearer the soil surface, if decomposition and dispersal had been allowed to take place over a longer period, and if the residues had been included in the soil sample used for assay, an increase in population density probably would have been detected. I have found that 4 or 6 days exposure of seed to soil moisture conditions too low for germination, as determined by Hunter and Erickson (50), did not adversely influence seed germination in sterilized soil, when the moisture content was raised; however, when Fusarium was present in the soil, emergence was reduced. Since Fusarium species are so com- monly isolated from plant residue (126, 127, 128, 129) soybean seeds (54, 58, 85, 101), and from the roots and rhizosphere of soybean (58, 88, 100, 103, 126), it is very likely that a sufficient Fusarium population would be present to reduce seed emergence in most soils if dry conditions per-~ sist for a few days. It is unlikely that matric potential values as low as ~15 bars would normally be present at planting. However, studies to determine the minimum soil moisture requirement for seed germination should not ignore the capability of Fusarium to rot soybean seeds. The possible role of Pythium in further reducing emergence of Fusarium~colonized seed is an- other consideration which should not be overlooked. PART IV ECOLOGY AND EPIDEMIOLOGY 0F PYTHIUM ULTIMUM PREEMERGENCE DAMPING-OFF 0F SOYBEANS INTRODUCTION Pythium Spp. have been well documented as plant pathogens (45) and are widely distributed in the continental United States (44). P. ultimum Trow, one of the primary pathogenic species on legumes, causing seed and root rot, has been thoroughly studied on hosts such as bean (Phaseolus vulgaris) (2, 29, 48, 67, 75, 89, 90, 91, 114) and peas (Pisum sativum) (36, 55, 56, 68, 69, 72), but substantially less is known about P. ultimum and soybean (Glycine max) (12, 13, 19, 53, 71, 106, 120). Most studies have shown that high soil moisture favors disease (36, 48, 56, 68, 91, 106), and temperature effects on disease often vary with the host (72). However, these studies have been conducted in the laboratory under controlled conditions and have not been verified in the field. Mathematical models used for predicting seed emergence have be~ come common in the literature and one has recently been developed for soybean (63). Models can be a very useful tool in identifying conditions in the field which limit seedling emergence. Such conditions can then be verified in the laboratory. This approach has not been used in study- ing soil-borne diseases of soybean. 53 54 The purpose of this study was to develop a multiple regression equation based on temperature, rainfall, and soil moisture data collected in the field to predict seedling emergence in soil naturally infested with P. ultimum. The influence of inoculum level, soil moisture and temperature on P. ultimum preemergence damping-off were also studied un- der laboratory conditions. MATERIALS AND METHODS Laboratorygexperiments: The Pythium ultimum isolate used was obtained from a rotted soy~ bean seedling and identified by F. F. Hendrix (Department of Plant Path- ology and Plant Genetics, University of Georgia, Athens, Georgia). Pythium cultures were maintained on V~8 agar in culture bottles at 5 C. The Pythium soil population was determined by a water agar method of Stanghellini and Hancock (113) which is useful for isolating and count- ing P. ultimum and other rapidly-growing species. Soybean pieces from which isolations were made were washed off under running tap water for approximately 30 min, then split in half and plated on water agar for isolation of Pythium and on Komada's Fusarium~selective medium (64, 65) for isolation of Fusarium. Water agar plates with drops of soil SUSpen- sion or which contained tissue pieces were incubated on the laboratory bench (23 2 2 C) in ambient light (5 to 8 lux during the day) unless otherwise indicated. The plates were observed after 24 and 48 hr. The use of Fusarium~selective medium is outlined in Part III. The soybean variety Hark was used throughout this study. Seeds used in laboratory experiments were disinfected as outlined in Part III. Seven seeds were planted on the surface of a 5 cm base layer of soil 55 56 (440 g of soil dry weight) in a plastic cup (14 cm X 11.5 cm) and then covered with approximately 3.8 cm of soil (400 g of soil dry weight). The cups were incubated in the dark and emergence was recorded 10 days after the time that 50% of the seedlings in disinfested soil emerged. The experiments were blocked (replicated) over time. In some experiments a soil moisture differential was established by watering the bottom soil layer on which the seeds were laid to ~0.013 bar matric potential and then covering with air-dry soil. 8y two days the soil moisture at seed placement was ~O.18 bar. Pots were watered daily from the bottom until the weight of the pot at planting was reached. A given matric potential was established in a soil sample by atomizing water onto dry soil in a plastic bag with frequent Shaking until a predetennined percent soil moisture level was reached which was based on the soil moisture matric potential curve (Fig. 4). In those experiments in which a constant soil moisture was tested, the pots were covered with a piece of plastic and placed in a plastic bag. The soil was a sandy loam whose characteristics are outlined in Part III. To effectively control Pythiaceous fungi, Lesan was added to the soil as outlined in Part III. Propylene oxide-treated soil (Part III) was infested by sprinkling an aqueous SUSpension of P. ultimum Sporangia from hemp seed broth cul- tures on the soil in a plastic bag (106). The bag was sealed and allowed to incubate at 23 2 2 C for a week after which it was air dried, sieved (4 mmz) and stored at 5 C. The soil population was determined before each set of experiments. Disinfested soil was added so that a given inoculum concentration was obtained. 57 Collection of field data: Multiple regression analysis was used to analyze data on soybean seed emergence which were taken from a field containing a high population (100-300 ppg) of P. ultimum. A treatment mean was made up of the average of two, 3 m rows each planted with 100 seeds. Seeds were sown at a depth of 6.5 cm unless otherwise indicated. During each experiment soil moisture, soil temperature and rainfall conditions were continually mon- itored. The data used for analysis were those recorded the first week of planting. Emergence was recorded 2 to 3 weeks after planting. Post~ emergence damping-off symptoms were rarely seen therefore percent emer- gence was felt to be a good disease index. To create different soil moisture levels, some rows were watered at the rate of 6.7-13.4 liters/row with a sprinkling can (roughly 1 to 2 cm of water) or watered heavily with 21.1-27.8 liters (3 to 4 cm). Rainfall was measured with a tipping bucket rain gauge (Weather Measure Corporation, Sacramento, Ca 95841); temperature was measured at a depth of 6.5 cm with a thermistor-probe de- signed for the Esterline Angus temperature recorder, ~20 to 1300 F (Es~ terline Angus Instrument Corporation, Indianapolis, Indiana 46224); soil moisture was measured with soil moisture blocks and the meter which was described in Part I. Soil moisture block calibration and field use: The soil moisture blocks were the gypsum cylindrical type (2.5 cm X 2.5 cm) with 15 foot leads obtained from Soilmoisture Equipment Cor~ poration, Santa Barbara, Ca. 93105. The blocks were calibrated in a ~15 bar pressure plate apparatus (Soilmoisture Equipment Corporation, Santa Barbara, Ca. 93105). A piece of nylon mesh (250 pm) was placed over the 58 plate, then a 0.5 cm layer of sieved (4 mmz) soil was added. The blocks were placed sideways on the plate and then additional soil was added to completely bury the blocks. Electrical connection with the blocks was made through an electrical lead-through assembly which was an extractor accessory. The soil and blocks were saturated overnight before applying the pressure. In the field, two moisture blocks were placed in one of the two rows at depths of 5.1 and 12.7 cm which were measured from the top of the blocks, unless otherwise indicated. A sandy-loam soil does not shrink and swell substantially enough to influence the block's contact with the soil. Multiple regression analysis: The multiple regression analysis was done by the stepwise dele- tion of variables method which is on the Michigan State University's statistical system computer program. The dependent variable was seed- ling emergence and no more than 5 independent variables were analyzed at one time. The program computes a regression equation containing all variables. The partial F test value is calculated for each independent variable treated as though it was the last variable to enter the re- gression equation. The variable with the lowest partial F is then eli- minated if it is less than the preselected Significance level of 0.10. The equation is then recomputed with one less variable and again the variable with the lowest partial F iS eliminated if less than the 0.10 level of Significance. The partial F criterion for each variable not in the regression at each step of calculation is evaluated again and in~ cluded if the partial F is greater than the 0.10 level (30). RESULTS Influence of inoculum and matricgpotential on emergence: As few as 25 propagules per gram (ppg) of P. ultimum in disin- fested soil completely eliminated emergence of soybean seedlings (Table 10). The influence of matric potential was tested using a moderate con~ centration of P. ultimum (7 ppg). At ~1.8 bars the emergence in soil containing P. uZtimum (66%) was not Significantly reduced compared to soil without Pytkium (73%) (Table 11). Even at ~0.18 bar matric poten- tial emergence (2 %) was not significantly (P_= 0.05) reduced below the control (65%) due to the large amount of variability. At ~0.018 bar the emergence was significantly (P_= 0.05) less, 15%, than that in disin- fested soil, 45%. The interaction between soil with and without Pythium and the moisture levels was not significant because even in the non- infested soil, emergence was reduced with increased moisture. Similar results were obtained with a naturally infested soil (Table 12). Emer- gence was significantly (P_= 0.05) less, 23%, at ~0.018 bar matric po~ tential than at ~0.18 (83%) or at ~1.8 bars (57%). The same trend was seen with other soil samples taken from the same farm. 59 60 Table 10. The effect of Pythium ultimum inoculum density in soil (~0.18 bar matric potential) on emergence of soybean seedlings at 28 C. Number of Percent propagules/g soil emergence 0 93:73 5 57 i 16 10 29 i 17 25 O aMean of 4 blocks of 7 seeds each. 61 Table 11. The influence of matric potential on soybean emergence in propylene-oxide treated soil with and without Pythium ultimum (7 propagules/g) at 28 C. Percent emergencea Matric potential ~bars with P. uZtimum without P. ultimum 0.018 15 ab 45 bc 0.18 29 b 65 DC 1.8 66 bc 73 C aMeans of 6 blocks of 7 seeds each. bMeans followed by the same letter are not significantly different from each other (P.= 0.05) using Tukey's w_procedure on transformed data: J x + 0.5 . 62 Table 12. The influence of soil matric potential values on soybean emergence in a naturally Pythium ultimum infested sandy-loam soil (500 propagules/g) at 28 C. Matric potential Percent a ~bars emergence 0.018 23 ab 0.18 83 b 1.8 57 b aMeans of 5 blocks of 7 seeds each. bMeans followed by the same letter are not significantly different from each other (P_= 0.05) using Tukey's w_procedure on transformed data: 7 x + 0.5 . 63 Influence of temperature on'emergence: To investigate the influence of temperature on disease develop~ ment, seeds were planted on wet soil and covered with dry soil. Under such conditions seeds germinated rapidly in disinfested soil and poorly if infested with P. ultimum. In natural soil with a Pythium concentra- tion of 500 ppg emergence was Significantly (P_= 0.05) higher at 16 C (23%) than at 20 (15%), 24 (13%) or 28 C (10%), and all values were less than the average of the disinfested soil over the four temperatures (96%). With another soil sample collected a year later which had a Pythium population of 300 ppg there was no significant (P_= 0.05) dif- ference between 16 and 28 C in soybean emergence (28 and 31%, respec- tively). At 16 C a Lesan soil treatment was included which increased emergence to 89%. When P. uZtimum (7 ppg) was added to propylene oxide- disinfested soil, a significant reduction in emergence occurred over a range of temperatures from 16 C to 28 C compared to soil at 28 C without Pythium (Table 13). The emergence at 16 C (9%) was significantly less than at 20 (44%), 24 (43%) and 28 C (60%). P. uZtimum reduced emergence in all the temperature studies but the influence of temperature varied with the soil sample. Investigating a possible Fusariam-Pythium interaction: Fusarium spp. were always present when Pythium was isolated from soybeans grown in natural soil (Table 14). Both fungi were easily iso- lated from the seed coats within 24 hr of planting. Pythium was not isolated from the tip of the primary root until 6 days after planting at which time it was only recovered from 30% of the pieces. Pythium could be easily isolated from cotyledons within one day and hypocotyls after 3 days. 64 Table 13. Effect of temperature on the emergence of soybeans in disin- fested soil infested with Pythium ultimum (7 propagules/g) and in non-infested soil. Temperature, C 552:3222e 16 9 aa 20 44 b 24 43 b 28 60 b 28 (control) 95 C aMean of 4 blocks of 7 seeds each. Seeds were planted in wet soil (~0.013 bar) and covered with air-dry soil. bMeans followed by the same letter are not significantly different from each other (P_= 0.05) using Tukey's w procedure on transformed data: x + O 65 Table 14. Percentages of soybean seedling tissue pieces from which Pythium and Fusarium spp. were isolated when planted in natural soil (300 ppg Pythium and 5,000 ppg Fusarium) at 28 C for 1, 3 and 6 days. b Day 1 Day 3 Day 6 Tissue d Pythiumc Fusarium Pythium Fusarium Pythium Fusarium Seed coats 84a 95 Root tips 0 O O 10 30 10 Cotyledons 21 7 6O 40 6O 6O Hypocotyls 33 20 70 60 aPercentages are based on 10 to 15 tissue pieces. bTissue pieces were from nonemerged plants and were approximately 4 mm2 in size. Lesioned tissue was plated when present. Seeds were planted in wet soil (~0.013 bar) and covered with air-dry soil (3.8 cm deep). CAll Pythium isolates grew at 10 C but none grew at 37 C. dF. oxyspom comprised 85% of the Fusamlum spp. isolated. 66 Three Fusarium isolates, F. oxysporum, F. monilifbrme 'subglu- tinans', and F. tricinctum, which caused seed and root rot of soybean in laboratory tests (105) were individually combined with Pythiam-infested soil (7 ppg) and adjusted to 3,000 ppg Fusarium. Seeds were laid on wet soil and covered with dry soil at 28 C. None of the Fusariwm isolates when added to soil decreased emergence below that of Pythium alone. Multiple regression analysis using_field data: The rainfall, soil moisture (matric potential) and soil tempera- ture data are shown in Figure 7. The soybean rows which were not arti~ ficially watered were used to form the regression equation to predict emergence because on those rows I had collected the most complete set of environmental data. The average percent emergence recorded for the nonartificially watered rows from May 17 to September 16 are Shown in Table 15 along with the predicted emergence based on the regression equation: y = 87.6-5.32(SM)~17.76(Rain) SM: The soil moisture variable (SM) was the number of days continuously after planting that the maximum soil matric potential at planting depth was >-0.5 bar plus the number of days in the first 3 days after planting that the matric potential at planting depth was <-3 bars. Rain: The rain variable (Rain) was the total rain- fall in cm within the first 3 days after planting plus the number of days in the first Figure 7. 67 Emergence of soybean seeds planted over the summer with corresponding data on rainfall, soil tempera- ture and soil moisture. The soil, 8 sandy-loam, was naturally infested with Pythium uZtimum. The emergence data were recorded 2 to 3 weeks after planting and are shown on the figure on the first day of planting. 68 MAXIMIN. SOIL MATIIC POTENTIAL I-IAISI m WT m WIT WT mm 5:. I3cun Sun 13cm 5‘. on u m a on u m a o— u m o o— u m u on u m a l l I I l 1 l __ ___________ _. \\~‘S\\\\\\\\\\\\\\\ x 4 4 4 \\\‘ \\\\\\\\\\\\\\\\’ N \\\\V . 4 \\‘~.\\\\\\\\\\\\\\\\ $\\\\\\\\V . S .\\\\\\\\\\\\\\\\- 4 1 4 4 4 .4 4 d 4 .4 -4 1 4 1 4 4 a S x\\\\\\\\\\\\\\\\\ x\\\\\\\\\\ 4 5‘" < 4 -I 4 II I or.) \\‘&\\\\\\\\\\\\\\ \\\‘§S\\\\\‘ 1 -( 4 $1 7 1 4 - 1 n: - a, -4 4 d 4 4 1 d 4 q 4 34 7. 3 b 3"» .1 1, a4 4 1 4418113584 I . 1'5 . I I I 1 J o T I I I T I I I j T T T n 3 s o z 3 ° 8 8 11 PERCENT EM EROENC E 69 Table 15. Actual emergence of soybeans planted over the summer, 1978, compared with predicted values based on a multiple regression equation. Planting Percent emergence Dates Observeda Predictedb May 17 57.5 56.0 May 26 75.0 87.6 June 3 83.5 71.6 June 8 51.5 71.6 June 20 93,0 84.9 July 4 81.5 61.0 July 12 79.0 70.8 July 27 47.0 61.0 Sept. 9 3.0 22.2 Sept. 16 0 5.5 aMean of two 3 m rows of 100 seeds each. bPredicted percent emergence (y) was based on the regression equation: y = 87.6-5.32(SM)~17.76(Rain). The soil moisture variable (SM) was the number of days continuously after planting that the maximum soil matric potential at planting depth was >~0.5 bar plus the number of days in the first 3 days after planting that the matric potential at planting depth was <~3 bars. Rain variable (Rain) was the total rainfall in cm within the first 3 days of planting plus the number of days in the first 3 days after planting that the matric potential at planting depth was <~3 bars. 7O 3 days after planting that the matric poten~ tial at planting depth was <~3 bars. The R2 for regression based on 10 plantings was 0.84 and the simple cor- relation between actual and observed emergence was (r_= 0.91). Data from 4 plantings of seeds at 3.5 cm depth, and which had corresponding moisture blocks with their tops at 2 cm depth had an r_= 0.74. Some of the 23 factors which were eliminated from the regression equation due to their low level of Significance are listed in Table 16. If the rainfall data are left out of the regression procedure a new prediction equation is formed: y = 79.03-6.64(SM) with R2 = 0.21. The emergence data ob~ tained from the watered rows correlated well with values from the Single value equation (r'= 0.72). The multiple regression analysis indicates that if a naturally infested soil is wet (>0.5 bar) or dry (<~3 bars) then emergence of soy- beans will be reduced. Rain in the first three days will reduce emer- gence further if the soil is not <~3 bars. 71 Table 16. A partial list of independent variables which were eliminated from the regression equation due to their low level of signi- ficance (>10%). 1) Weekly average soil temperature 2) Number of days maximum soil moisture was >~0.5 bar plus number of days soil moisture maximum was <~3 bars plus number of days average temperature was <25 3) Total rainfall 4) Rainfall the first day 5) Rainfall in the first 3 days 6) Average weekly soil moisture 7) Number of days soil moisture was >~0.5 bar 8) Number of hours of rain in the first week 9). Total rain in the first week DISCUSSION Even though P. ultimum is not known to produce zoospores (82), as few as 5 ppg of P. uZtimum in disinfested soil was found to signifi- cantly reduce soybean seedling emergence. No emergence occurred at 25 ppg. Pieczarka and Abawi (90) reported an 85% reduction in stand count of bean in wet soil that contained only one viable P. ultimum propagule/g oven-dry soil. They observed that increasing the population from 10 to 500 ppg made little difference in the amount of disease. Kerr (56), working with peas, found that it was necessary to take the cube root of the population of P. ultimum (O to 1083 ppg) in order to obtain a straight line plot against percent infection. The relationship between inoculum and disease often correlates poorly when field data are analyzed. Pieczarka and Abawi (90) sampled 8 bean fields in 1974 for root-rot severity caused by low temperature Pythium Spp. and found no correlation with population. They also found that the amount of variation in the Pythium population was nearly as great at a Single field sampling as that which occurred among the sample means from that field for the entire planting season. Adegbola and Hagedorn (2) tested 4 bean field soil samples under laboratory condi- tions and found that the number of Pythium propagules could not be con- sistently or significantly related to the amount of pea damping-off or Pythium bean blight. It appears that once a minimal population of a 72 73 pathogenic Pythium sp. is established in a field, further increases may be of minor importance compared to other factors in increased disease development (108). The Pythium population in soil may be relatively constant over a cropping season, or it may fluctuate. Hendrix and Powell (46) noted in one peach orchard that the Pythium population remained relatively con~ stant over 20 months whereas in another it varied over a 180-day period. The same type of variation was noted in two pea fields in Washington (69). If the population varied, it was usually lowest during periods of high temperature (75, 115, 130) or low soil moisture (97). Colonization of crop residue may also cause increases in the Pythium population (75, 117). V The emergence of soybean seedlings decreased with increased soil moisture in both P. uZtimum-infested soil and in propylene oxide- disinfested control soil. Emergence was least in the Pythium~infested soil. Increased disease severity with increased soil moisture has been consistently Shown for several Pythium spp. (7, 8, 18, 36, 38, 42, 48, 56, 68, 91, 98, 106). However, soil moisture is not always important in disease development (8, 40). The actual explanation for increased dis- ease with increased soil moisture can be very complex, as pointed out by Endo and Colt (33), because the presence of water may influence other soil and host properties such as oxygen and C02 levels, phytotoxic decomposi- tion products, host exudation, and host vigor. The influence of temperature on disease development varied de- pending on whether the soil was artificially infested or naturally in- fested. In soil which was inoculated with P. ultimum, most disease occurred at 16 C. This result agrees with what has been reported on 74 bean (91) and soybean (120). However, P. ultimum root rot of bean also has been reported to increase at high temperatures, 24 and 28 C, as com- pared to lower temperatures, 16 and 20 C (48). Their finding in arti- ficially infested soil agrees with my results obtained in naturally in- fested soil where seedling emergence was greatest at 16 C. P. uZtimum blight of snap bean (2) and root-rot of peas (68) have also been reported to increase with higher temperatures. In another field soil sample, I found no difference in soybean emergence between 16 and 28 C, which is Similar to a report on P. ultimum preemergence rot of safflower (60) and seedling rot of clover (41). The temperature effect on disease is prob~ ably related more to its influences on the host than on the pathogen (72). There appears to be no interaction between Fusarium spp. and P. ultimum in causing preemergence damping-off of soybean seedling under moist soil conditions. This conclusion is based on the facts that Lesan (Dexon) treatment of the soil restored emergence, and that Fusarium spp. did not increase disease when added to P. ultimum soil. Dunleavy (31) reported that F. oxysporwm may cause poor emergence of soybean in moist soil; however, I was never able to verify his findings. Interactions have been reported between P. ultimum and Fusarium spp. (55, 89), and it is very possible that Pythium and Fusarium spp. may interact as root rot pathogens once the soybean is beyond the seedling stage. Mathematical models can be a valuable tool in identifying en- vironmental factors which are favorable or deleterious to the crop. Hypocotyl elongation rate, which correlates highly with emergence (61), was measured in the field by Knittle, Burris and Erbach (63) and used in a regression equation to identify limiting factors for soybean emergence. During the gennination phase of the seed they found that soil moisture 75 correlated positively with subsequent hypocotyl elongation. However, during the elongation phase of the seedling devel0pment, soil moisture and soil resistance to penetration were highly negatively correlated, but soil temperature was highly positively correlated with emergence. Regression equations Should not be used indiscriminately because each soybean variety or seed lot may respond differently to environmental factors (14, 32, 51, 61, 62, 63, 79, 102, 131, 132). From analysis of my field data with multiple regression, I found that the number of days of continuously wet soil (>~0.5 bar) from the time of planting, plus the number of days of low soil moisture (<~3 bars), positively correlated with disease. It is not known whether Pythium causes poor emergence in the field soil at matric potentials <~3 bars, but Pythium has been known to colonize tissue at low poten~ tials (66). Seedlings germinate very slowly at ~3 bars, and may thereby remain susceptible longer to infection by Pythium and perhaps Fusarium spp. Since rainfall during the three days immediately after planting reduced emergence, it is very likely that the higher the soil matric potential in the field the more disease will develop. Gypsum soil mois- ture blocks are not very accurate at potentials >~0.5 bar; therefore, this value was used as the lower limit in the analysis. AS pointed out by Knittle and colleagues (63) in their regression equation, wet soil may reduce soybean emergence irrespective of the presence of a pathogen. I was not able to develop any independent variable involving tem- perature data which was statistically significant. The temperature may not have been important because of the narrow range of weekly average temperatures which were recorded, the lowest average being 20.3 and the highest 28.2 C. 76 The multiple regression equation which was devised is by no means a complete model for the disease because factors such as seed vigor, seed size, soil type, planting depth, soybean cultivar, and inoculum density of the pathogen were not included. However, through the use of multiple regression techniques the importance of soil moisture in P. uZtimum damping-off has been verified. 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