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THE ROLE OF SOIL-HORNE PUNGI IN THE DRY BEAN ROOT ROT COMPLEX BY Maureen Ford Mulligan A THESIS submitted to Michigan State University in partial fulfillment of the requirement for the degree of MASTER OF SCIENCE Department of Botany and Plant Pathology 1983 (3 race/.2 ABSTRACT THE ROLE OF SOIL-BORNE FUNGI IN THE DRY BEAN ROOT ROT COMPLEX BY Maureen Ford Mulligan The data suggests that excessive tillage practices may in- crease dry bean root rot by affecting bulk density, percent porosity and water drainage of saturated soils. Although disease severity was inversely correlated with several parameters of plant productivity, final yield did not correlate with disease severity or tillage treatments. Estimations of root rot severity indicated that a threshold infection point was determined above which yield was reduced and below which, yield was not affected. Excessive tillage treatments decreased mycorrhizal colonization as did depth. Bean cultivars with vigorous root systems main- tained high levels of mycorrhizae deep in the soil profile. Data from pot experiments indicated that when beans are grown under well-watered conditions Fusarium solani f. phaseoli may have de- trimental effects on the ability of the host plant to take up water. In addition, the pathogen may alter trichomes on bean hypocotyls to facilitate root penetration. ACKNOWLEDGEMENTS I would like to thank Dr. Gene Safir for the privilege of working in his laboratory, and for the support and friendship he has provided me. I also would like to thank those members of my committee, Dr. A. Smucker, Dr. A. Saettler and Dr. L. P. Hart for their efforts and helpful suggestions. This thesis would not have been possible without the help of my fellow collegues. In particular I would like to thank R. Michael Mulligan of the Biochemistry Department, and Nicholas Bolgiano, Lynn Epstein, Vicki Dunevitz, Annette Tumolo, Helen Kuhn and Janet Keeney of the Botany and Plant Pathology Department. Excellent technical assistance was provided by Peggy Wilbur and Eric Hall. ii LIST OF TABLES TABLE OF CONTENTS LIST OF FIGURES Introduction Chapter I. Chapter II. Chapter III. Chapter IV. Bibliography The Effect Of Three Tillage Practices in Two Fields on Dry Bean Root Rot and Yield. Materials and Methods Results Discussion Mycorrhizal Colonization of Dry Bean Root Systems Grown in Compacted Soils Materials and Methods Results A Discussion The Effects of Dry Bean Root Rotting Fungi on the Host Plant Water Relations Materials and Methods Results Discussion An Additional Mode of Dry Bean Root Penetration by Fusarium solani f. phaseoli Materials and Methods Results Discussion iii Page iv 21 22 25 36 4O 41 44 50 54 54 57 65 68 69 71 76 78 LIST OF TABLES Table ~ Page 1. Stages of development of legumes. 25 2. Bulk density and percentage porosity for 26 three tillage practices. 3. Root rot ratings (expressed as the % of root 32 area infected) for four dry bean cultivars grown on Charity clay soil averaged over all tillage treatments. 4. The effects of tillage on yield and root rot 35 ratings for four dry bean cultivars grown on Charity clay soil. 5. Yield parameters and root rot ratings for four 35 dry bean cultivars grown on Charity clay soil averaged over all tillage treatments. 6. Effects of type and available soil phosphorus 45 on mycorrhizal infection of Black Turtle Soup grown on conventionally tilled soils. 7. The effects of tillage on mycorrhizal infection, 45 plant growth and tissue phosphorus for Black Turtle Soup grown on Charity clay soil. 8. The effect of soil depth on mycorrhizal 50 colonization of three dry bean cultivars grown on a Hillsdale loam with conventional tillage. 9. Diffusive resistance values measured by a 65 diffusion porometer and calculated from an equation using and Ohm's law analogue. iv Figure 1. 10. ll. 12. LIST OF FIGURES Page Soil moisture characteristic curves for two 27 soil types subjected to three secondary tillage treatments. Comparisons of the standard root rot index 28 scale and the percentage of ropt area infected scale plotted against the grams dry weight per plant for all secondary tillage treatments on the sandy loam soil. Disease developmental curves for cultivar 30 Black Turtle Soup grown on Belleville sandy loam. Disease developmental curves for cultivar 31 Black Turtle Soup grown on Charity clay. The effects of root rot 65 days after 34 planting on dry bean yield components grown on Charity clay. The effects of depth on soil bulk density 47 for three secondary tillage treatments on Belleville sandy loam and Charity clay. The effects of depth on mycorrhizal 48 colonization for three secondary tillage treatments on Belleville sandy loam and Charity clay. Mycorrhizal developmental curves for cultivar 49 Black Turtle Soup. Equilibration curves for the Wescor dewpoint 59 hygrometer at low and high water potentials. Relationship of leaaf water potential values 60 between dewpoint hygrometer and the pressure bomb for excised dry bean leaf tissue. Measured and calculated water relation . 62 parameters of well-watered dry bean plants. Relationship between leaf diffusive 64 resistance and leaf water potential for the cultivar Swan Valley. 13-16. Scanning electron micrographs of Swan 73 Valley hypocotyl segments with early root rot symptoms. 17-19. Scanning electron micrographs of Swan 75 Valley hypocotyl segments with early root rot symptoms and control plant. vi Introduction Dry beans (Phaseolus vulgaris L.) are worldwide in distribution and constitute one of the most important food crops of the world. Michigan is a leader of edible dry bean production in the United States and, this industry contributes substantially to Michigan's agricultural income. The yield per acre is a very important factor affecting the profitability of crop productiOn; therefore, declining yields due to fungal, bacterial and virus infestations are very serious problems. Root rot may cause significant re- ductions in dry bean yields in Michigan and is usually associated with soil stress conditions such as compaction and inadequate soil moisture. Interactions of pathogens with other soil microorganisms can significantly alter the severity of root disease. Mycorrhizal fungi have been shown to offer protection against root rotting fungi for a large number of plants and it is possible that they could affect the dry bean root rot complex as well. The role that environmental factors and soil borne fungi have in the dry bean root rot complex are reviewed here. Root rot, caused by a complex of pathogens, can be a significant factor contributing to the declining bean yields. Dry bean root rot was first recognized in 1916 in western New York by Burkholder (1916). Its occurrence has been reported on many different bean varieties throughout the 0.5. and in foreign countries (Abbott 1929, Kovachevsky 1931, Ruokola 1978). Fusarium solani (Mart.) Appel and Wr. f. sp. phaseoli (Burk.) Snyd. and Hans. is thought to be the primary causal agent in the Michigan dry bean ”root rot com- .plex'. Thielaviopsis basicola Zoph. and Rhizoctonia solani Kuhn are of secondary importance (Wyse 1973). In New York soils, Pieczarka and Abawi (1978) observed that bean root rot was more severe when Pythium ultimum Trow. and F. solani were present together. No interaction was observed between 3; solani and g; solani, but Rhizoctonia reduced the severity of root rot caused with Pythium gpé. Steadman et a1. (1975) reported that beans in Nebraska were infected by R; solani early in the season and then predisposed to infec- tion by the main root rotting pathogen, g; solani by the sixth week. Pfender and Hagedorn (1982) have reported 2; ultimum and Aphanomyces euteiches (Drechs.) f.sp. phaseoli Pfend. and flag. are the major causal agents of bean root rot in Wisconsin soils. These latter pathogens have not been found to be of importance in the root rot complex in Michigan and therefore will be omitted in further discuss- ions. 5; solani f.sp. phaseoli is highly pathogenic to roots and stems of Phaseolus vulgaris L. Infected roots and hypo- cotyls initially develop small red-brown lesions which eventually coalesce into larger lesions extending to the soil line (Burkholder 1919). The red color is replaced by brown discoloration and frequently accompanied by longitudi- nal fissures which, in many cases, can destroy the taproot. Small lateral roots which normally develop from the taproot can be killed. Such lateral and taproot destruction is then followed by proliferation of fibrous adventitious roots near the soil surface. If soil moisture levels are excessive or too low, diseased plants are stunted. If adverse conditions persist, the leaves turn yellow and yield reductions may be severe. On the other hand, if the fibrous, secondary roots are not disturbed and adequate moisture and nutrients are a- vailable, the effects of root rot may be minimal. The fungus is believed to be introduced into an area on infested seed or in infested soil (Nash and Snyder 1964). It is also believed to accumulate with repeated cropping of beans (Menzies 1952). Using a plate count method, Nash and Snyder (1962) discovered a uniform distribution of the bean root rotting Fusarium population, in a field recently cropped to beans. The population of the pathogen decreased after beans were not planted for 1.5 years. Vertical dis- tribution of F. solani f. phaseoli propagules coincided with the distribution of bean roots. Also, tillage distributed propagules throughout the plowed layer and propagules were rarely isolated from soils below the plow zone (Burke et al. 1972, Burke 1969). It is interesting to note however, that fields with the highest propagule numbers did not necessarily have the worst infection (Nash and Snyder 1962, Burke 1966). In greenhouse studies, Burke (1965b) noted that dissemination of the pathogen was limited to splashing water droplets on the soil surface. Infection did not spread to the non-infected parts on the same plant or to neighboring plants. Chlamydospores are thought to be the primary inoculum in field soils. Macroconidia placed in field soil convert directly into chlamydospores or germinate via a short germ tube and then produce chlamydospores (Nash et a1. 1961). The fungus also produces chlamydospores within the cortical tissue of infected bean roots (Burkholder 1919). In contrast, Maloy (1960) reports that the mycelial form of the pathogen predominates in the soil. When the host tissue is not available, the pathogen has been reported to have a~ limited saprophytic growth and can perpetuate itself in a soil environment free of susceptible plants (Nash et a1. 1961, Schroth and Hendrix 1962). This was also suggested by Schroth and Snyder (1961) and Cook and Schroth (1965) who reported that g; solani f. phaseoli chlamydospores germinated when various amino acids and sugars were added to the soil. Common amino acids and sugars were used to simulate non-host root exudates, which may be capable of providing limited nutrients for saprophytic growth. These authors also reported that chlamydospores germinated when placed near root tips or germinating bean seeds; however, 5 root exudate from mature bean roots did not stimulate germination. Cook and Snyder (1965) found 60% of the chlamydospores germinated when adjacent to seeds of g; vulgari . The germ tubes were lysed quickly by other micro- organisms unless susceptible host tissue was available. This suggests that limited saprophytic growth is not a major factor in inoculum production. Many reports are available on the mode of penetration and host-parasite relations of g; solani f. phaseoli (Christou and Snyder 1962, Chatterjee 1958, Burkholder 1919, Bywater 1959). Most researchers agree that initial infections occur through stomata in the hypocotyls. The hyphae also penetrate the roots and infect either directly through the epidermis or through mechanical or natural wounds. Wounds formed by the emergence of the lateral roots are a common site for penetration. Infrequently, hyphae can enter through the bases of damaged or dead root hairs. Burke and Barker (1966) determined tap root and hypocotyl disease may be incidental to more important damage which occurs in the lateral root system. The data indicated the need to control Fusarium root rot of beans by treatments which protect the lateral root system as well as the taproot and hypocotyl. The bean root rot pathogen typically forms a thallus on the host surface before penetrating host tissue (Christou and Snyder 1962). Glucose was essential for hypocotyl penetration and nitrogen stimulated pathogenesis and penetration (Toussoun et al. 1960). Maurer and Baker (1965) observed organic nitrogen was more effective then inorganic nitrogen in stimulating both the development of the thallus on the hypocotyls and the expression of symptoms. In con-4 trast to this, Papavizas et al. (1968) showed that the abil- ity of the nitrogen form to influence root rot depended on the soil type and pH. The dry bean root rot disease occurs most often when the plants are subjected to conditions unfavorable for optimum growth. Bean roots are particularly sensitive to many environmental and cultural practices that predispose them to infection by the root rotting fungus (Burke et a1. 1980). Soil compaction, occurring as a result of current season tillage, to be an important factor in the Fusarium root rot problem. Plants have limited root growth as a result of compaction (Schumacher and Smucker 1981) and ex- perience water stress in years when rainfall is deficient. In addition, excessive water, or flooding, often causes com- pacted soils to become oxygen deficient. As moisture is re- moved by the transpiring surfaces of plants a potential _ gradient is established from the leaf to the root and into the soil. Since water moves in the soil by bulk or mass flow, and depends on pore size, changes in the soil moisture content have marked changes on the hydraulic conductivity of water. Water which remains in the smaller pores and as thin films around the soil particles is largely unavailable to plants. Excessive tillage decreases the large pores in the soil and results in an increase of tiny pores which causes water to drain at higher suctions possibly favoring the pathogen. Fusarium stalk rot of sorghum was reduced in Nebraska by use of minimum or no-tillage practices (Doupnik et al. 1975). The reduced tillage methods did not prevent dry bean root rot infection but permitted the development of deeper more vigorous roots than those grown in compacted soils. Widely spaced plants have less root rot then those growing closer together. The beneficial effects of wide spacing are thought to be due to the increase of plant growth and lower plant competition; however, this decrease in disease severity is nullified if soil temperatures are low (Burke 1965a). Less than optimal soil temperatures reduced the ability of bean roots to penetrate compacted soils more than loose soils. Conversely, loose soils and temperatures favorable for rapid plant growth reduce root rot severity (Burke 1965a, Burke and Nelson 1965, Burke et a1. 1980). A combination of cultural control methods which reduce soil compaction and provide appropiate irrigation have been successful in controlling the disease. While typical severe Fusarium root rot ocurred in all field plots, subsoiling treatments done directly before planting promoted root development and significantly increased yields (Burke 1968, Burke et a1. 1972, Miller and Burke 1974, Natti 1963). Subsoiling followed by plowing or discing was not beneficial. Yield increases appeared to result from greater regeneration of roots and increased rooting depth and volume, rather than reduced infection. While plants growing in the non-subsoiled field plots were small and water stressed between irrigation times, plants in subsoiled plots were larger and did not show stress between irrigations. When optimal soil moisture levels were maintained, the plant yields were similar in infested and non-infested soils, regardless of subsoiling treatment. Subsoiling combined with sprinkler irrigation was more effective in reducing root rot than subsoiling and rill irrigation. Root rot severity is also increased by excessive irrigation. Bean roots were severely injured by Fusarium gp; when subjected to short periods of near zero oxygen levels caused by excessive wetting in the soil and, in oxygen depleted soil, roots could not penetrate the deeper soil profiles. However, excess wetting had little effect on bean growth in non-infested soils (Miller and Burke 1975). Bean roots resistant to root rot in well-aerated soils became susceptible to disease when exposed to brief periods of oxygen stress in the presence of the pathogen. Soil compaction, resulting from excessive tillage and traffic, and the concomitant anoxia, mechanical impedance and reduced availability of water and nutrients, generally restricts growth of the plant root systems (Schumacher and Smucker 1981). The detrimental effects of soil compaction, water stress and Fusarium root rot are additive and, when found simultaneously in the field cause significant yield losses (Miller and Burke 1974). In the presence of decreased soil water, plants with infested roots cannot penetrate the compacted layers in the soil, whereas plants grown in fumigated soil can penetrate the compacted soil zone. Plant damage was greatest when plants were grown in Fusarium-infested, compacted soils at low water potentials. Conversely, yields were highest in non-infested, non-compacted soils maintained at optimal water content. Matrix potentials influence the amount of bean seed ex- udation and hence the activity of g; solani f. phaseoli and g; ultimum (Stanghellini and Hancock 1971). Reid (1974) provides evidence that both the quantity and quality of exudates change with changes in substrate water potential. In view of the importance of root exudates on the colonization of roots by microorganisms (Bowen and Rovira 1976), water stress induced changes in root exudation may also have marked effects on host penetration. There are water potentials which are optimal and minimal for the growth of every microorganism. Water potentials for optimal growth can differ with temperature (Bruehl et a1. 1971) and pathogens can also respond 10 differently to osmotic versus matric potentials (Adebayo and Harris 1971, Cook et a1. 1972). Many researchers have demonstrated the requirements for growth and reproduction of fungi (Adebayo and Harris 1971, Cook et a1. 1972, Sommers et a1. 1970, Duniway 1975, MacDonald and Duniway 1978, MacDonald and Duniway 1978b). Cook and Papendick (1970) published a review on the effects of soil water on microor- ganisms and their relation to soil-borne fungal diseases of plants. However, information on the influence of water potential for the reproduction and growth of Fusaria species is limited. Macroconidial production by Fusarium roseum Lk. emend. Snyder and Hansen (Graminearum) and Culmorum was highest at about -15 bars matric potential and prevented at -50 to -60 bars. Mycelial growth by these isolates was best at a matric water potential (-10 bars) which was similar to the optimum for conidial production. However, growth of mycelium was prevented at water potentials of -100 bars, a value much lower than that limiting the production of conidia. In addition, when salts were added to the soil, hyphal growth was increased as the osmotic potential was lowered down to -20 bars. This growth stimulation at the lowered water contents has been attributed to ion uptake by the hyphal cells. Sommers et al. (1970) suggest that the reduced fungal growth rate in matric systems may be related to an inability of the fungus to absorb solutes and thus osmoregulate in dry soil. A simple explanation for the 11 " increase of disease severity during low soil water conditions is that hyphal growth of the pathogens may be stimulated. Even after infection, fungal growth would be subject to the hosts' internal water status and thus, water stress may favor the development of Fusaria in the plant via lower water potentials (Cook and Papendick 1972). In general, plants subjected to water stress have a larger incidence and greater severity of disease. For example, withholding water from safflower before inoculation with Phytophthora cryptogea Pethyb. and Laff., increased the severity of root rot (Duniway 1977b) and charcoal stalk rot of sorghum plants occurred only after onset of stress conditions (Odvody and Dunkle 1979). Foot rot of wheat increased in severity only when densely planted wheat was treated with high amounts of nitrogen and then subjected to water stress (Papendick and Cook 1974). The water potentials of the plants dropped during water stress and ex- tensive culm decay occurred; however, at low planting density, water potential of the plants remained nearly constant during water stress (even under high nitrogen) and disease development slowed down. Hence, with adequate available soil water, disease could be kept under control. This can also be said for dry bean root rot in that there is a greater incidence of root rot in fields with narrow rows than in fields with wide rows (Burke 1965). 12 It is well established that plant water stress can reduce photosynthesis (Boyer 1976) and impede downward location of photosynthate. Some root diseases cause stomatal closing after an increase in leaf water stress. Some pathogens produce chemicals which directly inhibit sto- matal opening. In contrast, one or two diseases cause abnormally large stomatal openings and excessive transpira- tion (Creatura et a1. 1981) which would lead to dessication and tissue death under some conditions. Working with Fusarium wilt of tomato, Duniway (1971) reported that xylem resistance to water flow was much higher in diseased plants than in healthy plants. The leaves of diseased and healthy plants wilted at similar water potential values and diffusive resistances showed that stomates reacted similarly to water stress. The results indicated that the wilting was solely a product of the high xylem resistance (Duniway 1971), and not a product of pathogen produced toxins (Gaumann 1958, Gottlieb 1944). Similarly, Phytophthora root rot of safflower caused leaf wilt at the same water potential as water stress in healthy plants. Wilting was reversible for both diseased and healthy plants (Duniway 1971, 1973). Duniway concluded that the water relations of wilting involved in the Phytophthora root rot of safflower are similar to the water relations of wilting in Fusarium wilt of tomato and Verticillium wilt in cotton. Interestingly, the main resistances to water flow 13 in safflower were located in parts of the stem and internodes where the pathogen was not found (Duniway 1977). Sugar beet plants infected with Aphanomyces cochlioides Drechs. showed a significant increase in resistance to liquid water flow and in diffusive resistance of leaves to vapor flow (Safir and Schneider 1976). Due to the increase of flow resistance, diseased plants had less water available and consequently, decreased transpiration rates. Several workers have postulated that the incorporation of plant residues into soil, would induce the biological control of Fusarium root rot of beans. Theoretically, an organic amendment rich in carbon and low in nitrogen stimulates the growth of many different microorganisms. Such a highly competitive environment is not conducive to the growth and reproduction of g; solani f. phaseoli (Snyder et al. 1959). When lignin and chitin were added to soil, a slight decrease in disease resulted; however, addition of glucose nullified this control. This suggested carbon was required for pathogenicity and that competition among competing microbes reduced disease. In addition, it has been observed that immature residues of rye and corn with a high C:N ratio stimulated disease severity but mature res- idues reduced disease. Immature rye and corn-amended soils had enough nutrients present (anthrone and ninhydrin positive materials) to allow chlamydospore germination. With the absence of nutrients in the mature rye and 14 corn-amended soils, germination did not occur (Maurer and Baker 1964, Lewis and Papavizas 1977). Bandara and Wood (1978) reported that organic fractions isolated from sterile soil filtrates contained compounds that both inhibited and stimulated chlamydospore germination. There has been very little work to actually test the effects of plant residue incorporation into Soil in the field.* Nash and Snyder (1962) studied the effect of rotation crops on bean root rot populations. They found that when barley rotations followed bean there was an increase in Fusarium propagules whereas tomato rotations appeared to cause a decline in the population. When garlic extracts were incorporated into the field before planting, foot rot of g; vulgaris was controlled. Lower concentra- tions of garlic extracts were proportionately less effective (Russell and Mussa 1977). Biological control of disease with mycorrhizal fungi seems promising but only a few studies have been done in the field. Several observations suggest mycorrhizal associations can greatly improve plant growth and nutrition (Mosse 1973, Marks and Kozlowske 1973, Sanders et al. 1975). Numerous reviews on this subject are available (Reid and Bowen 1979, Safir 1980, Hayman 1982). Theoretically, improved plant nutrition and water relations would increase plant vigor and thus the host would be more resistant to pathogen attack. However, Dehne (1982) recently published a 15 review on the interaction of mycorrhizae with plant pathogens. Mycorrhizal plants apparently withstand the stress of infection from root pathogens better than their non-mycorrhizal counterparts (Schonbeck and Dehne 1977, Zambolim Nemec 1979, Zambolim and Schenck 1982-personnal communication). Mycorrhizal plants were larger even though the roots of both mycorrhizal and non-mycorrhizal plants had the same degree of disease symptoms. Chou and Schmitthenner (1974) reported that fewer soybeans were killed by Phytophthora megasperma Drechs. var. ggjag Hildeb. in Enf dogone-treated soil than in soil without Endogone. The severity of the root rot was not affected by g; mosseae (currently classified as Glomus mosseae) which indicated that the symbiont did not dispose the host to infection or enhance the intensity of the disease. Nemec (1979) suggests that the vesicular-arbuscular mycorrhizal fungi are effect- ive symbionts of both diseased and healthy trees. Working with citrus trees infected with blight, he observed that the overall mycorrhizal infection was similar for both the diseased and healthy plants. Since the blighted trees had a reduced root capacity he assumed that there was a direct reduction in the effective complement of the mycorrhizal symbiont. Similarly, mycorrhizal fungi have been shown to increase papaya shoot height but decrease root weight in the presence of Phytophthora palmivora (Schenck and Kellem 1978). Here, the vesicular-arbuscular mycorrhizal fungus 16 decreased the root volume which was detrimental to the plant while increasing shoot height which was beneficial to the host. Ross (1970, 1972) previously showed that chlamydospores of the mycorrhizal fungus Endogone s2; stimulated soybean growth and yields yet, stem rot symptoms were enhanced in the susceptible cultivars by the addition of the mycorrhizal fungus. However, the mycorrhizal fungus had no effect on stem rot severity in the resistant cultivars. A similar type of synergistic-antagonistic relationship has also been seen on cotton plants, where increased shoot growth due to mycorrhizal benefits resulted in an increase in the incidence of Verticillium dahliae. In this case, however, the scientists observed that the disease severity was the same in non-mycorrhizal plants when they applied high amounts of phosphorus fertilizer (Davis et a1 1979). The increase in foliar disease of mycorrhizal plants may thus be due to the increased nutrient status of the plants and their higher physiological activities. The increased metabolism of the host cell cytoplasm is apparantly an advantageous en- vironment for plant viruses. For example, plant virus titer has been clearly demonstrated to increase in mycorrhizal plants versus non-mycorrhizal hosts (Daft and Okusanya 1973). Schenck and Kinloch (1974) discovered an inverse correlation between the incidence of endomycorrhizal fungi 17 and populations of the root knot (Meloidogyne incognita) and cyst nematodes (Heterodera glycines Ichinoke). The nematode population decreased when the mycorrhizal population increased. This antagonistic effect was noted at low nematode levels for the susceptible variety and at the high nematode levels on the resistant variety. Safir (1968) found lowered percent infection of onion by Pyrenochaeta terrestris in the presence of vesicular-arbuscular mycor- rhizal infection. Becker (1977) further characterized this situation in that mycorrhizal onion root segments were more resistant to the pathogen (P. terrestris) then the non-mycorrhizal segments of the same root system. These data suggest that increased mycorrhizal infection will in- crease pink root disease tolerance in onions. There are several reviews on the effects of environ- mental factors and mycorrhizae (Reid 1979, Bowen 1980, Safir and Nelsen 1981). It has been suggested that mycorrhizae are advantageous to the host plant under moisture stress conditions (Safir et al. 1971, 1972, Nelsen and Safir 1982, Sanders et a1. 1975). Soybeans inoculated with vesicular- arbuscular mycorrhizae increased shoot growth and exhibited less resistance to water flow then the non-mycorrhizal control plants. The calculated difference in hydraulic conductance between mycorrhizal and non-mycorrhizal soybeans was attributed to the improved nutrient status of the plants associated with the symbionts (Safir et a1. 1971, 1972). 18 This was clearly demonstrated when non-mycorrhizal soybeans were fertilized with high amounts of phosphorus and the resistance to liquid water flow was equivalent to the mycorrhizal plants. In accord with this data, Levy and Krikun (1980) observed that no difference occurred in the hydraulic conductivity of mycorrhizal citrus plants versus non-mycorrhizal plants grown under a high nutritional regime (daily watering with 0.1% solution of 20:20:20, N:P:K fertilizer). The better recovery from water stress by my- corrhizal citrus seedlings was apparently related to effects on stomatal regulation. Safir and Duniway (1982) reveiwed other environmental factors which influence vesicular-arbuscular mycorrhizae. Schenck and Schroder (1974) noted a reduction in root growth of mycorrhizal soybeans compared to non-mycorrhizal soybeans when plants were grown at sub-optimal temperatures (18 C); however, at optimal temperatures myccorrhizal fungi stimu- lated growth. Reid and Bowen (1979) observed that excessive soil moisture conditions resulting in anaerobiosis reduces the growth and infection by mycorrhizal fungi. The authors ,also suggest that low soil water potentials decrease mycor- rhizal colonization. I found that compacted soils affected the colonization of vesicular-arbuscular mycorrhizae. In the non-compacted soils mycorrhizal colonization was well developed by the time the plants were setting flowers. Thus it appears that mycorrhizal fungi were established and able 19 to benefit host development when a large proportion of the total nutrient uptake needed to occur. In the compacted soils, sparse colonization of roots was seen at mid-season indicating an extended lag phase (Sutton 1973) and hence, probably precluded any significant influence on host nutri- tion. Evidence indicates that the nitrogen status of legumes is enhanced by vesicular-arbuscular mycorrhizae which indirectly affects nitrogen fixation by its action on phosphate uptake (Daft and El-Giahmi 1974, 1975). The -stimu1ated plant growth of mycorrhizal plants has also been related to a net increase of photosynthetic activity in the plants. Hayman (personal communication) suggests that this increase is due to the higher chlorophyll content of mycor- rhizal plants. Other researchers have reported on the in- creased levels of growth regulating hormones. Both ectomy- corrhizae and vesicular-arbuscular mycorrhizae have been shown to increase the gr of hormones called cytokinins. These hormones are known to influence numerous aspects of plant growth including ion transport, photosynthetic rates, transpiration rates, and root growth (Christensen and Allen 1979, 1980, Miller 1967). Also, Baltruschat and Schonbeck (1975) reported an increase in arginine levels due to vesic- ular-arbuscular mycorrhizae. The experiments done in this study concentrate on the role environmental factors have on infection of dry bean roots by pathogenic and symbiotic fungi. Field studies done 20 in the dry bean regions of Michigan, examined the effects that tillage practices had on the structure of the soil and severity of Fusarium root rot. Using cultivars bred to withstand compaction and root rot, I wanted to find out if the cultivation methods used by Michigan bean growers were aggravating root rot and, if so, was this affecting their economic yields. Sutton (1973) demonstrated that patterns in host development have been related to mycorrhizal devel- opment. Since plant growth is stunted in compacted soils, then colonization of the symbiont may also be diminished. The effects of tillage practices on mycorrhizal colonization was also examined. Pot experiments were set up to characterize the effects Fusarium solani f. sp. phaseoli had on the water relations of the host plant. An examination of the mode of penetra- tion by the pathogen was also studied with the use of the. scanning electron microscope. The data will be used for a yield reduction systems model being developed in our lab. 21 Chapter I. The Effect 2; Three Tillage Practices 13 Two Fields 93 Dry Bean Root Rot and Yield. Root rot, caused by a complex of pathogens, can be a significant factor contributing to declining bean yields. Dry bean root rot was first recognized in western New York by Burkholder (1916). Its occurrence has been reported on many different bean varieties throughout the U.S. and in foreign countries (Abbott 1929, Kovachevsky 1931, Ruokola 1978). Fusarium solani (Mart.) Appel and Wr. f.sp. phaseoli (Burk.) Snyd. and Hans. appears to be the primary causal agent in the Michigan dry bean "root rot complex" (Wyse 1973). It is highly pathogenic to the root and stem of Phaseolus vulgaris L. Infected roots and hypocotyls initi- ally develop small red-brown lesions which eventually co- alesce into larger lesions extending to the soil line (Burkholder 1919). The red color is replaced by brown dis- coloration and frequently accompanied by longitudinal fis- sures which in many cases, can destroy the taproot. Small lateral roots which normally develop from the taproot can be killed. Such lateral and taproot destruction is then followed by proliferation of fibrous adventitious roots near the soil surface. If soil moisture conditions are excessive or too low, diseased plants are stunted. If adverse condi- tions persist, the leaves turn yellow and yield reductions 22 may be severe. On the other hand, if the fibrous, secondary roots are not disturbed and adequate moisture and nutrients are available, the effects of root rot may be minimal. To develop rational and economical control measures, the magnitude of yield loss due to root rot must be evalu- ated and related to the gain obtained. Only by disease-loss appraisal is it possible to determine economic loss due to different degrees of disease. During the 1981 growing season, field experiments were conducted to characterize the relationship between disease and loss in yield. A more com- plete understanding of the timing of infection as it per- tains to soil compaction and soil moisture was obtained for a dry bean yield reduction Systems model being developed in our lab. Disease progress curves were developed as well as a method for estimating yield loss for a given amount of disease. Materials 32g Methods During the 1981 growing season, field plots were estab- lished (as part of a larger study designed to measure plant growth and yield responses to soil compaction) at two sites (on different soil types: a Charity clay (Michigan State Uni- versity Bean and Beet Research Farm, Saginaw, Michigan) and a Belleville sandy loam (Bay County, Michigan). Three secondary tillage treatments were applied at both sites which had received primary moldboard tillage and were: 1) minimal tillage - no secondary tillage in the spring, 2) 23 conventional tillage - a secondary tillage done with a tandum spring tooth and spike tooth harrow in the spring be- fore planting and, 3) excessive tillage -a secondary tillage. (as previously noted) plus one complete pass with a sheep foot roller and wheel to wheel tractor track (four passes) coverage. Field plots were banded with fertilizer 5.1 centimeters deep and 3.8 centimeters to the side according to the recommendation of the Michigan State University Ex- tension Service. Seeds were planted on May 28th and June 4th, 1981, for the Belleville sandy loam and Charity clay sites, respectively. The experimental design was a com- pletely randomized block; blocking against the moisture gradients in the fields. Each treatment was replicated five times. The experimental unit was four rows (2 meters) by 18 meters. All sampling was taken from the middle two rows in each replicate. Soil samples were taken according to the core methods described by Blake (1965), to determine the bulk density and soil porosity for each tillage treatment. These undisturbed soil core samples were also used to determine the functional relation between soil wetness and matric potential in the low suction (-0.01 to -0.06 bars) range, by means of a pressure plate assembly. Swan Valley, NEP-Z, Black Turtle Soup (BTS), and Domino were the dry bean varieties planted at the Charity clay plots while BTS and Seafarer were the bean varieties planted 24 at the Belleville sandy loam soil site. All varieties were sampled three times throughout the growing season. The three sampling times were an early vegetative, anthesis and pod filling stages at 30, 45, and 65 days after planting. Bean roots were sampled from the upper soil profile and root disease ratings included the total area of root plus hypo- cotyl, seven inches directly below the cotyledons. Hypo- cotyl was defined as that portion of the stem below the cotyledons and above the main tap root. Disease and yield‘ were the two main variables recorded at each sampling date. Disease assessment included disease incidence (number of plants infected expressed as a percentage of the total num- ber of plants assessed) and disease severity (the area of plant tissue affected by disease). To obtain the most sensitive reading of disease for modeling purposes, disease severity was expressed in two ways: A) scale of 0 to 4 using a standard root rot index where; (0) no infected tissue, (1) discolored tissue over 20 to 50% of the hypocotyl, (2) dis- colored tissue over 50 to 75% of the hypocotyl with 20% or less of the area covered with lesions, (3) primary root com- pletely discolored with 20% or more lesions; some secondary root development, (4) primary root completely rotted with or without secondary root development and B) estimating the percentage of the root area infected (7 inches below the bean cotyledons). The weighted average of 20-25 plants was used per disease reading. Whenever disease and yield 25 parameters were recorded, the growth stage of the crop was also noted (Table 1) so that meaningful comparisons could be made. Table 1. Stages of development of legumes Major stages Substages 1 Vegetative 1 Early - 4-6 inches high 2 Medium - over 6 inches high 3 Late - pre-bud (few stems in pre bud stage) 2 Bud 1 Early - buds minute, may be felt as an enlargement in apex of stem 2 Medium - buds well formed and visible 3 Late - buds visible, swollen: earliest buds showing some color at tips 1 10% bloom 2 25% bloom 3 4 1 2 1 2 3 3 Flower 50% bloom 75% bloom 100% bloom Flowers dying Early - green pods Medium - seed in soft stage Mature - seed mature 4 Full flower 5 Seed (After a system developed by Dr. J. E. Winch, University of Guelph) Results Excessive tillage practices significantly affected the bulk densities of both soil types and consequently the per- centage of total air pore space (Table 2). Soil moisture drainage under saturated conditions was also limited by the compacted soils. The relationship be- tween soil water potential and moisture content of the two different soils and three tillage practices is shown in 26 Figure 1. Note the gradual decrease in water potential with decreased percent soil water and vice versa. The saturated water content and the initial decrease of water content with the application of low suction are reduced with the excess- ive tillage. The soils subjected to minimal tillage treat- ments and the conventional tillage treatments appeared to hold more water than did the soils subjected to excessive tillage treatments at these high water potentials. At approximately -0.05 bars water potential, soil water content was equivalent for all three secondary tillage treatments in the sandy loam soil. In the clay soil, essentially no water drained from the compacted soils and decreased water poten- tials were necessary to make the water drainage equivalent for all three tillages. Table 2. Bulk density and percentage porosity for three tillage practices. Charity Clay Belleville sandy loam Tillage B.D. (glcc) 3 porosity B.D. (g/cc) ‘3 porosity minimal 1.16 55.4 1.56 40.7 conventional 1.22 53.8 1.58 40.0 excessive 1.40* 47.0* 1.67* 36.9* * Values in each column are significantly different at P=0.05 according to Duncan's multiple range test. The incidence of disease was 98% in each bean field for the 1981 growing season. A comparison between the standard root rot scale and the percentage scale is illustrated in PERCENT SOIL MOISTURE ‘27 Du ID .0; V’ ID— to M 6—1 a, m- N m 53‘ swunrtouu [.3- g— 10.7 z: mthmlflkne ‘ouwuflumiflinp o cmnuhwiflqp O F f f I F l 0.00 0.01 0.02 0.03 0.04 0.06 0.06 HRTER POTENTIRL (-BRRSJ Figure 1. Soil moisture characteristic curves for two soil types subjected to three second- ary tillage treatments. -28 ET 2. : 1 9", o 9‘ I ' . 9 ° '1 . 81 . .‘d . o- ‘ . z 0 g . . : :8 . .0 f i C K , ; o . f. I ° . . . 5:. : al: 0. o ; . . : 5i . c.. . a I . . . . O 0 52+ ‘ I 8 -°l..° .'5 :°.- . (i . 8‘ z . o. o. e 1 . . : : : o B .‘ 2 z .4 ... o 0' B . B 2 ': !.! o . z I . o ‘ . o .. ’0‘. i ° 0 .0... . g 0 . o = 0“ o '. £0. '0' ' . ° 0 H L l : .v‘ ” ‘8: O o- . I . ,° ;} 38‘13 . , . 5' .' a ' . 5 a g a .5 a ; .5 6 R007 .6? WIN-7 Figure 2. Comparisons of the standard root rot index scale and the percentage of root area infected scale plotted against the grams dry weight per plant for all secondary tillage treatments on the sandy loam soil. Readings were taken 65 days after planting. 29 Figure 2 for the sandy loam soil. It shows the relation be- tween disease severity and yield. The percentage scale seem to be more sensitive. This scale illustrates a threshhold point of disease after which yield was affected. The yield of the host plant was affected when greater than 35-40% of the root surface is damaged by the fungus. Below this critical point, plants can apparently compensate for the loss of root growth with consequently no loss in yield. Each point on both scales represents grams dry weight of in- dividual-plants versus disease severity. Although variation in sampling was high, trends of the depressing effects on yield due to root rot can be seen. Due to the high varia- . tion of the individual readings, the weighted average of 20-25 plants per reading was used for future analyses. Excessive secondary tillage practices increased the severity of root rot of dry beans grown on clay and sandy loam soils. Figure 3 and Figure 4 illustrate the develop- ment of disease for the variety BTS for the three secondary tillage practices. They show the development of root rot of this cultivar grown on the sandy loam (Figure 3) and Charity clay (Figure 4) soils subjected to the three seconary till- age treatments. Each point is the mean of 5 replications and represents the average disease reading from approximate- ly 100 plants. Significant root rot differences (P-0.05) resulting from excessive tillage were recorded 45-65 days after planting. Cultivar differences also began to occur at 30 to— a) m— at .F- CD a: '5 a‘ C3 0: .— 2: ur*- 0 v-0 0: DJ 0. &flhMMe b" sandy loom 0 mums: '? umwnfimdlfl a. quintet” o i F I l I 0 15 30 45 60 75 DFl‘tS HFTER PLHNTING Figure 3. Disease developmental curves for cultivar Black Turtle Soup grown on Belleville sandy loam. 31 ID— on BTS 85-23.? m... N p. CD a: I— "“ o N CD a: p. I: u:*- c)" , 3J1 Choruty Clay 0. r~d Clwdwhulfll QLInmthmdll fir «nu-hell c, I I r T I 0 15 30 45 60 75 DRYS FIFTER PLFINTING Figure 4. Disease developmental curves for cultivar Black Turtle Soup grown on Charity clay. 32 this time (Table 3). Table 3. Root rot ratings (expressed as the % of root area infected) for four dry bean cultivars grown on Charity clay soil averaged over all tillage treat- ments. Days Afterilanting Cultivars 39 25 65 BTS 21.3la 24.39b* 28.98b NEP-Z 22.55a 23.30ab 26.023 SWAN VALLEY 21.60a 24.59b 29.60b DOMINO 19.723 21.03a 27.09a * Values for eaCh column followed by the same letter are not significantly different according to Duncan's multiple range test (P-0.05). Bean cultivars predisposed to damage or destruction by the Fusarium root rot complex at bud break had depressed yields during the growing season (Figure 5); however, dis- ease ratings were low and when compared with final yields there was no effect due to tillage nor differences amongst varieties (Table 4). This was true for both soil sites. Final yields were not affected by tillage treatments or dis- ease severity. Hence, even though disease severity was in- creased by tillage practices and this increase in disease affected plant growth throughout the growing season (Figure 5), the plants were able to compensate by some means (e.g. number of pods per meter, Table 5). 33 Figure 5. The effects of root rot 65 days after plant- ing on dry bean yield components grown on Charity clay. # SEED/POD SEED/POD G./NETER POD NT. 7‘ 11 21 2:3 PERCENT ROOT ROT I 35 POD Wt Figure 5. T T I I r 7 14 21 28 35 PERCENT ROOT ROT G./HETER SEED NT. G-/HETER DRY NT. ID :— §- SEE WT. ID 2“- O' x 3‘ x X ”—1 l‘ M O:- IO M IO N l T I T r. O 7 14 21 28 35 PERCENT ROOT ROT D N- 0" 8 (0" lflOflEUD 8‘ X N X O (0-! N O X a“ x 534 X N D 8 l T T T I— 0 7 14 21 28 35 PERCENT ROOT ROT 35 Table 4. The effects of tillage on yield and root rot ratings for dry bean cultivars grown on Charity clay soil. Tillage firybean gATields % Root rot % Root rot cultivar kg/ha (45 days) (65 days) Minimal Black TurtIe Soup 2902abc* 22.83abcd 27.6855 Swan Valley 3002abc 21.61abcd 28.07bcd NEP-Z 3115c 20.69abc 23.40a Domino 3038bc 17.65a 27.18ab Conventional Black Turtle Soup 2851ab 21.39abcd 28.29bcd Swan Valley 2805ab 25.11bcde 28.68bcd NEP-2 2932abc 25.06bcde 25.35ab Domino 2762a 19.47ab 24.10ab Excessive Black Turtle Soup 28l7ab 28.95e 30.96cd Swan Valley 2793ab ‘27.04de 32.04d NEP-2 2888abc 24.16bcde 29.31bcd Domino 3046bc 26.25cde 29.90cd * Valuesifollowed by the same letter in each column are not significantly different according to Duncan's multiple range test (P-0.05). Table 5. Yield parameters and root rot ratings for 4 dry. bean cultivars grown on Charity clay soil averaged over all tillage treatments. Parameters Domino Neg-2 BIS Swan Valley % Root Rot 21.13a* 23.30ab 24.39b 24.59b # Seed/Pod 5.9a 5.3b 5.0bc 4.7c #Pods/meter 266.7b 287.0ab 295.9ab 320.3a Seed wt.g./100 19.07a 18.33ab 17.73bc 17.13c Yield kg/ha 2949a 2978a 2859a 2867a * Values going across the row follcwed by the same letter are not significantly different according to Duncan's multiple range test (P-0.05). 36 Discussion This paper demonstrated that excessive tillage practices may increase disease severity by affecting bulk density, percent porosity and decreasing water drainage of saturated soils. Although disease severity was inversely correlated with several parameters of plant productivity (Figure 5), final yield did not correlate with disease severity for the cultivars tested. 'The cultivars tested have been bred to withstand compaction and subsequently root rot. Atypical climatic conditions in the fall (1981) pro- vided a lengthened growing season and optimal soil moisture. These conditions may have extended the root growth which compensated for the deleterious effects of root rot on the plant water status and compensated for growth losses which occurred earlier in the growing season. It was not until 45 days after planting that signif-. icant differences in disease severity due to tillage were recorded (Figure 3 and Figure 4) and inverse correlations with bioyields (total dry weight of plant) obtained (Figure 5). Bagget and Frazier (1973) also reported this to be the case in Oregon bean fields. Hence, I am suggesting that root rot ratings taken before anthesis (45 days after plant- ing) may not be of major significance for predicting yield losses. When comparing the severity of root rot with final yields (Table 4) no correlation was observed. 37 Since bulk density includes both pore spaces and solid particles, the excessive secondary tillage treatments re- sulted in compacted soils with higher bulk densities due to decreased pore space. Those soils in which compactipn was minimized were looser and more porous and thus had lower bulk density values. The physical changes associated with plowing and cultivating are structural rather then textural. Although soil texture is of great importance in determining soil characteristics, the structure of the soil also influences soil parameters such as water movement, aeration, bulk density and porosity (Brady 1974). Soil moisture retention in the high water potential range (0-1 bar) is strongly in- fluenced by soil structure and pore size distribution (Hillel 1972). Compaction reduces the number of macropores and increases the proportion of small and medium-sized pores, which tend to hold the water with a greater suction than do large pores (Brady 1974). As I have shown, changes in the soil structure, due to excessive tillage, influenced the relationship between percent soil moisture and soil water potential (Figure 1). This indicates a reduction in the soil volume contributed by the macropores. Bulk water movement also occurs through the macropores and is decreased under these conditions by almost 5-10 percent (Figure 1). This decrease in soil moisture may be of significance to the the growth of crop plants. In addition, compacted soils may 38 " become anaerobic, and this may predispose bean roots to root rotting pathogens (Miller and Burke 1975). Poorly drained soils result in low oxygen diffusion rates due to the low oxygen content in the soil air or high soil water contents. Miller and Burke (1977) concluded that Fusarium root rot was aggravated by low oxygen diffusion rates in saturated soil. Soil compaction substantially alters the environment surrounding plant roots. Mechanical impedance also reduces growth and modifies the morphology of roots growing in com- pacted soils (Russell 1977). Several investigations have demonstrated the effects of soil compaction on the dry bean root rot problem (Burke 1968, Burke et al. 1962, Miller and Burke 1974). I have also demonstrated the difference in disease levels due to minimal and conventional tillage ~methods versus excessive tillage methods. In addition, Doupnik et al. (1975) reported that Fusarium stalk rot of sorghum was reduced in Nebraska by use of minimum or no-till practices. One explanation for these observations could be that mechanical impedance induces changes in the rbot sys- tem, predisposing them to oxygen deficiency. The percentage scale has many advantages over the root rot index for modeling purposes (Figure 2) as suggested by James (1974) for other systems. The upper and lower limits of the percentage scale are well defined: 0-100, the scale can be divided and subdivided conviently, it is universally known, incidence and severity are expressed on the same 39 scale (James 1974), and most important, it gives a more sensitive reading of infection. The fact that a critical point may exist on the percentage scale (between 35-40 percent of the root area infected), above which yield may be affected, is an additional advantage over the standard root rot index and has not been demonstrated by other research- ers. The importance of this is shown in Table 4. When com- paring the severity of root rot with final yields no correl- ation was observed. This suggests that 1) the percentage of root area diseased was too low during the 1981 growing sea- son and hence, below the critical point on the scale and 2) plants with disease ratings below the critical point were able to compensate for the degree of root rot. For example the number of pods per meter (Table 5) increased as disease severity increased. A universally known method such as the percentage scale may provide adequate standardization and consistency of disease measurment plus the possibility of determining economic loss. 40 Chapter II. Mycorrhizal Colonization 2; Dry Bean Root Systems Grown ig_Compacted 801 s. Vesicular-arbuscular mycorrhizae are among the most common fungi in the soil environment. They infect most species of agricultural crops in most habitats (Gerdeman 1968). However, various agricultural practices, particu- larly fertilization, can significantly affect mycorrhizal root colonization and spore production (Hayman 1982). Generally high soil fertility is considered to lead to little mycorrhizal infection so that it is unlikely to find crops heavily mycorrhizal in intensively cultivated soils. Soil compaction, resulting from excessive tillage and traffic, and the concomitant anoxia, mechanical impedance and reduced availability of water and nutrients, generally restricts the growth of plant root systems (Schumacher and Smucker 1981). Hence, one could assume that these negative effects could ultimately increase mycorrhizal colonization. However, Sutton (1973) demonstrated a distinct 3-phase se- quence of mycorrhizal development over time where the highest rate of colonization coincided with the host's greatest demand for nutrients. Since patterns in host development have been related to mycorrhizal development and plant growth is considerably stunted in compacted soils then, colonization of the symbiont may be diminished. To our knowledge, there are no published reports in- volving specific studies of the relationship between tillage 41 practices and mycorrhizal infection. This study was de- signed to examine the influence of multiple tillage practices on mycorrhizal infection in dry bean plants. Since compaction has been reported to increase the severity of root rot in dry beans (Burke et al. 1972, Miller and Burke 1974), a comparison between root rot resistant and susceptible varieties and mycorrhizal colonization was also evaluated. Methods and Materials Field studies were established on three soil types at three locations in the dry bean production area of Michigan. Several dry bean varieties were grown on a clay soil (Charity series) in Saginaw County, sandy loam soil (Belle- ville series) in Bay County and loam soil (Hillsdale series) in Ingham County. Primary tillage was applied as a mold- board plow. Three secondary tillage treatments were em- ployed at the Charity clay and Belleville sandy loam plots. Tillage treatments were: 1) minimal tillage - no secondary tillage treatments, 2) conventional tillage - four secondary tillage passes with a tandum spring tooth and spike tooth harrow and 3) excessive tillage- excessive secondary tillage and traffic which included four passes with a sheep's foot roller, wheel to wheel tractor traffic plus four passes with a tandum spring tooth and spike tooth harrow. The Hillsdale loam site had only a conventional tillage treatment. 42 Field plots were banded with fertilizer 5.1 centimeters deep and 3.8 centimeters to the side according to the re- commendation of the Michigan State University Extension Ser- vice. Seeds were planted on May 28th, June 4th, and June 6th, 1981, for the Belleville sandy loam, Charity clay and Hillsdale loam sites, respectively. The experimental design was a completely randomized block; blocking against the moisture gradients in the fields. Each treatment was repli- cated three times. The experimental unit was four rows (2 meters) by 18 meters. All sampling was taken from the middle two rows in each rep. The upper 15 centimeters of the soil profile were sur- veyed at germination for indigenous populations of mycorrhizal spores (Glomus gpy). Phosphorus levels in the soils were analyzed at this time by the Bray 1 method. Plant tissue phosphorus contents were measured with the plasma emission spectrophotometer throughout the season. Bean roots were sampled 45 days after planting when the plants were in a preflowering stage. Vertical root core samples were taken according to the methods of Srivastava et al. (1982) to the depth of 48 centimeters at all three soil locations. This entire sample was then subsampled into cubes by a fractionating cutter giving six depths of eight centimeters (444 cubic centimeters) per depth. Bulk densities for all the tillage treatments were determined at all six depths. In addition, bean roots were also sampled 43 from the upper 10 centimeters of the soil profile and rated for mycorrhizal infection three times throughout the growing season. The 3 sampling times were: an early vegetative, preflowering and pod filling stages at 30, 45, and 65 days after planting. These samples were used to develop mycor- rhizal developmental curves. Roots were separated from the soil by the hydropneu- matic elutriation method (Smucker et a1. 1982). Cleaned roots were stored in small plastic bags and preserved in 10% formaldehyde until the degree and intensity of mycorrhizal development was evaluated. Cleaned roots were stained using the method modified from Philips and Hayman (1970). Six root segment subsamples per treatment were mounted on a glass slide and rated for intensity of infection as described by Nelsen et a1. (1981) using a scale from 0 to 4 where: (0) no evidence of infection, (1) observed only entry points, (2) small areas of hyphae occupying less than 5% of the segment, (3) hyphae present throughout the section at low levels or heavily concentrated in less than one-half the root, and (4) hyphae and vesicles concentrated throughout the entire root segment. Bean varieties used were NEP-Z, Black Turtle Soup (BTS) and Seafarer. NEP-Z and BTS have been bred to withstand soil compaction and root rot. Seafarer has less vigorous roots and is susceptible to compaction and root rot. All three cultivars were assessed for mycorrhizal infection on 44 the Hillsdale loam plot. Mycorrhizal infection of roots was determined at six depths in the soil profile. Black Turtle Soup and Seafarer were assessed on the Belleville sandy loam site and BTS was assessed at the Charity clay soil experi- ments. Results Mycorrhizal inoculum levels before planting were approximately two spores (Glomus sp.) per gram of dry soil. The distribution in the upper 15 centimeters of the soil profile was equal at each location. The conventional tillage treatment was used for comparison of infection between locations. Even though the number of mycorrhizal spores were equal at all locations, the intensity of infection was significantly less at the Belleville sandy loam site (Table 6). The Hillsdale loam soil had the highest amount of mycorrhizae infection and the Belleville sandy loam the lowest. The soil with the highest soil P, Belleville sandy loam, had the lowest mycorrhizal infection. " 45. Table 6. Effects of type and available soil phosphorus on mycorrhizal infection of Black Turtle Soup grown on conventionally tilled soils. Soil Typg Soil P Myc. rating (k . a.) Belleville sandy loam 441a 1.96s Charity clay 59b 2.32b Hillsdale loam 112b 2.66b * Values in each column followed by the same letter are not significantly different according to Duncan's multiple range test (P-0.05). Mycorrhizal colonization (45 days after planting) was reduced due to excessive tillage. Also, plant growth in the compacted soil treatments was lower than plants grown in the less compacted soils (Table 7). High mycorrhizal ratings had a direct relationship with plant size and both of these parameters were not correlated with tissue phosphorus levels. Table 7. The effects of tillage on mycorrhizal infection, plant growth and tissue phosphorus for Black Turtle Soup grown on Charity clay soil. Mycorrhizal Dry weight Tissue Tillage Rating g/meter Phosphorus , (PPM) Minimal tillage 2.60a* 97.34a .316ab Conventional tillage 2.32a 97.14a .282a Excessive tillage 1.79b 37.25b .345b * Values in each column followed by the same letter are not significantly different according to Duncan's multiple range test (P-0.05). 46 Secondary tillage and traffic increased the bulk densi- ties of the soils in the Belleville sandy loam and Charity clay soil sites (Figure 6). Soil bulk densities were simi- lar below the plowline for all three tillage treatments. Greater soil compaction due to excessive tillage and traffic appeared to decrease mycorrhizal colonization. Also, 'mycorrhizal infection was inversely related to soil depth (Figure 7). Using root samples taken from the upper 10 centimeters of the soil profile, mycorrhizal developmental curves were produced (Figure 8). A significant decrease in infection (P-0.10) in the compacted soils was noticed at the preflow- ering stage but not earlier or later in the season. Relatively high levels of colonization were observed in the roots of all treatments at the pod filling stage. Excessive tillage and traffic appeared to reduce the rate of mycorrhi- zal colonization of the dry bean cultivar BTS. All three dry bean cultivars grown on the Hillsdale loam were heavily colonized with mycorrhizae in the upper levels of the soil profile (Table 8). No difference existed between cultivars down to the plowline (32 centimeters). However, NEP-Z and BTS maintained significantly higher levels of mycorrhizal infection below 32 centimeters. The high amount of mycorrhizal colonization in the less com- pacted upper soils was comparable to the high levels found in the lower, more compacted soils (Table 8). Seafarer, the BULK DENSITY (G/CC) BULK DENSITY (G/CC) Figure 47 .4 "2 "1 CHARITY CLAY ’ '3“ l n n .1 l 0 en I 2.. a I ‘3 .P'°"'" " l 5:- ' 8 . ..'.“ n ate-alive tillage —— $0.97 to 4 conventional Glace -—-—-n-o.92 '2- f 0 minimal tlllage $0.97! an 3 L .. N ‘7 :27 animus SANDY LOAN “2.1 D “3.4 d' "3- O * 0-l N * :4 g I excessive tillage R="O.12ne .1‘ +conventicnal tillage R2039." o 0 minimal tillage 880.77 C0 .2 r r r T r T I 0 8 18 24 32 40 48 56 TlFPTH i CM . 6. The effects of depth on doil bulk density for three secondary tillage treatments on Belleville sandy loam and Charity clay. NS standsfor not significant at P=0.05. l 3 1 l (NYCORRHIZRL INFECTION 2 48 Charity clay plowline 3! excessive tillage “ll-82 4 conventional tillage ='0-95 0 minimal tillage R=‘0-97 2 Bellwllle sandy loam o ' ! excessive tillage n=-o.85. : 9 conventional tillage Ian-0,97 Q 0 minimal tillage Rat-0.92 uJ u. .J z m g—n 5% ha “[4 H J: O: ‘3 .1 L) " ' )— I: o r r r T r r I 0 8 16 24 32 40 48 56 DEPTH l Cl‘l . 1 Figure 7. The effects of depth on mycorrhizal colon- ization for three secondary tillage treat- ments on Belleville sandy loam and Charity cla . Allylines are significant at P=0.05. -49 z '4 Belleville sandy loam o 0—0 p. L) m . ll... 0‘) z “' shfius .1 spans a «i 4 H / :I: (K abflW5 a: - I . o .. ‘T (.1 > 0 minimal tillage z o conventlpnal tillage I n “’15 so 45 so DRYS RFTER PLFlNTING Figure 8. Mycorrhizal developmental curves for culti- var Black Turtle Soup. 50 susceptible cultivar to root rot and soil compaction was un- able to maintain high levels of mycorrhizae in the roots deep in the soil profile. Table 8. The effect of soil depth on mycorrhizal colonization of three dry bean cultivars grown on a Hillsdale loam with conventional tillage. fiepth (centimeters) Cultivar 8 lg 33. 32 ‘49 28 Nep-2 3.1ab* 2.9abc 2.8abcd 2.5bcd 2.5bcd 2.3bcde BTS 2.6bcd 2.9abc 3.1ab 3.0abc 2.5bcd 1.8de Seafarer 3.6a 3.0abc 2.8abcd 2.0cde 1.4ef 0.8f * Values followed by the same letter are not significantly differeet according to Duncan's multiple range test P=0.05 . Discussion We have shown that tillage practices may affect mycor- rhizal colonization and development in dry bean roots. These results appear to be the first report of the relation between tillage and compaction and mycorrhizal infection. Mycorrhizal colonization of dry bean root systems grow- ing on three levels of tillage (Figure 8) was similar to the three-phase development pattern for vesicular-arbuscular mycorrhizae reported by Sutton (1973). The lag phase, al- though not reported here, appears to occur during the first 21 days in greenhouse studies (unpublished data), the phase of extensive mycorrhizal development appeared to occur up to day 45 with the constant phase occurring after 45 days. It 51 should be noted that these relative developmental phases were modified by secondary soil tillage. The causes for which excessive tillage practices may decrease mycorrhizal colonization of dry bean roots are unknown. It is known; however, that compacted soils result in smaller, 1ess vigorous root systems. Thus one may assume that a smaller root volume comes in contact with a smaller volume of inoculum. On the other hand, the well aerated soils result in larger, healthier root systems which are able to grow throughout the rhizosphere increasing its chances of coming in contact with mycorrhizae fungal propa- gules. In the non-compacted soils, mycorrhizal relation- ships were greater by the time the plants were setting flow- ers (45 days after planting). Thus it appears that mycor- rhizal colonization was established in time to benefit host development when a large proportion of the total nutrient uptake occurred. In the compacted soils; however, a smaller amount of root colonization was seen at the preflowering stage indicating an extended lag phase and hence, probably precluding any significant influence on host nutrition. Sanders (1975) and Menge et a1. (1978) demonstrated the effects of high plant phosphorus content in the control of mycorrhizal infection. These studies showed that high soil phosphorus levels did not inhibit infection if plant phos- phorus was low. Although, decreased mycorrhizal infection on the compacted soils may be due to the diminished growth 52 rates (Table 7) I did not observe an inverse correlation with increased levels of tissue phosphorus in the plants. It has also been thought that soil water status can play an important role in mycorrhizal infections. Reid and Bowen (1979) observed that excessive soil moisture condi- tions resulting in anaerobiosis reduces the growth and in- fection by mycorrhizal fungi. The authors also suggest that low soil water potentials can decrease mycorrhizal infec- tion. Both of these soil moisture conditions are often found in the compacted soils of Michigan bean fields. Ex- cessive rains which cause flooding often cause compacted soils to become oxygen deficient. In addition, compacted soils provide less available soil moisture for plant growth due to the distribution of pore sizes. The increase of small pores due to excessive tillage results in water drain- age at higher suctions thereby making water less available to the plant roots and surrounding microflora. As roots grow deeper into the soil profile they enter more compacted soils below the plow line. Mycorrhizal inoculum levels also drop below the plow line (Sutton and Barron 1972) and therefore, one may speculate that the decrease in mycorrhizal infection in the deep profile samples (Figure 7) is due to the increase in compaction com- bined with the decrease of mycorrhizal inoculum and root mass. This is supported by the reports of Sutton and Barron (1972) and Sutton (1973). 53 Of special significance, is the demonstration that those varieties with vigorous root systems can penetrate compacted soils below the plow line and may carry with them mycorrhizal benefits of increased nutrient absorption. Burke (1969) reported that bean root rot propagules are sel- dom found below the plow zone. Therefore, the vigorous root systems of NEP-2 and BTS were able to escape the presence of the pathogen by penetrating the deeper, more compacted layers of the soil profile yet still remain mycorrhizal. Damage to the root from the outside by the development of the bean cortical root rotting fungi may destroy the foodbase for mycorrhizal fungi in the living root tissue. However, if resistant varieties possess vigorous root systems that can penetrate the compacted soils below plow lines or grow in a compensatory manner in less compacted areas, and carry_with them mycorrhizal benefits, the plant may be able to escape the pathogen and derive the advantages of the symbiont. 54 Chapter III. Egg géégcgséfii ggyggage¥§%%ofigtting Eggs; 25 Moisture stress in beans is a principle problem caused by root rot; however, when optimal soil moisture is provided to root rotted beans, yields are similar for diseased and healthy plants (Burke 1965, Miller and Burke 1974). Root diseases have been shown to cause a significant increase in resistance to liquid water flow and in diffusive resistance of leaves to vapor flow (Duniway 1971, Safir and Schneider 1976). Due to the increased resistances, the authors con- cluded that diseased plants have less water available re- sulting in stomatal closure and consequently, decreased transpiration rates. Data are not available on the relationship between host water potential and root rot pathogenesis of drybeans. In this report we characterize the effects of the bean root rotting fungus Fusarium solani (Mart.) Appel and Wr. f. sp. phaseoli (Burke.) Snyd. and Hans. on several host water re- lation parameters. Methods 33g Materials The seeds of two dry bean cultivars, moderately resis- tant, NEP-Z and moderately susceptible, Swan Valley, were germinated in vermiculite. The seedlings were transferred to Styrofoam cups containing equal amounts of Belleville sandy loam soil. The soil was sieved through a 2mm screen and autoclaved at 15 p.s.i. for one hour and allowed to cool 55 overnight before planting. Immediately prior to transplant- ing the root of each plant was dipped in distilled water or 100 ml of fungal inoculum. The inoculum was prepared from a single spared culture of Fusarium solani f. phaseoli (Bay County isolate) and diluted to a concentration of 106macro- conidia per ml. Plants were grown in a growth chamber with a 14 hour photoperiod, air temperatures of 26°C (dey)/18°C (night) and a relative humidity controlled at a 65 1 10 per- cent level. Pots were arranged in the growth chamber in a complete- ly randomized design. All plants were grown under well-watered conditions and there were 4 replicates per treatment. After 16 days all pots were watered to a common weight and measurements of all parameters made. All experi- ments were performed twice. Pots were wrapped in aluminum foil and then enclosed in plastic wrap to eliminate evaporation from the soil or pot. Pots were weighed twice during the light period and once during the dark period. Transpiration was measured by dividing the amount of water loss by the average of the leaf surface area. Leaf surface area was determined at the end of the experiment with a Li-Cor portable leaf area meter (model LI-3000). Leaf water potentials were measured using the Wescor dewpoint hygrometer and C-52 sample chambers following a previously described method (Nelsen et a1. 1978). Leaf 56 discs were excised from the middle leaflet of the youngest, fully expanded trifoliate, placed into the sample chambers in the growth chamber and allowed to equilibrate. Relative diffusive resistances of individual leaves were determined with a Li-Car diffusion parameter (model LI-65). The measurements were taken under well-watered conditions during the middle of the light period directly before sampling for water potential measurements. Stamatal resistances ta vapor flow were also calculated using an Ohm's law analogy and transpiratianal data. The calculated whale diffusive resis- tance for the whole plant served as a good calibration tech- nique far the parameter. Diffusive resistance was calcul- ated using an equation described by Kramer (1969), leafr+ airr= leafVE- airv X 0.622 Eq. 1 T where: rsleaf and boundary layer resistance to vapor flaw in s/cm vpsvapar pressure in mmHg T=transpiratian rate in cm3/cm2/sec 0.622=canstant of density of air:atmaspheric pressure g/ml/mmHg Leaf vapor pressure (leafvp) was determined by measur- ing leaf temperature with an Omega Digicatar Model 406 and assuming the turgid leaf was at 100% relative humidity. Air vapor pressure (airvp) was determined using a dewpoint 57 hygrometer (Yellow Springs Instrument Ca.). The boundary layer resistance (0.41 sec/cm) was estimated from the vapor lost from wet filter paper cut into the shape of a drybean leaf (Nelsen and Safir 1982). The resistance is zero for wet filter paper and since there is no cuticle, the only‘rev sistance to vapor loss is the boundary layer resistance. The leaf resistance was determined by subtracting the value calculated for boundary layer resistance (0.41 sec/cm) from the value obtained in Equation 1 for leaf plus boundary lay- er resistance. The ease with which liquid water flows through the plant is represented as the hydraulic conductivity (K) and it is the inverse of plant hydraulic resistance. It was calculated according to the equation: K 3 Eq. 2 T Iq'L. as) where: T is the transpiration rate at a steady state, 5 is the water potential of soil (assumed to be negligible be- cause experiments were performed with moist soil and there- fore given a value of 0 bars)(Boyer 1971), and L is the water potential (bars) of the leaf surface. Results The water potential of all samples was measured after a three hour equilibration period (Figure 9). In addition, a 58 comparison was made between the Wescor dewpoint hygrometer and a pressure bomb designed for the field (Sail Moisture Equipment Corp. Model 3000), to see if consistent and acceptable methods for determining leaf water status could be made in the field as well as the lab. The hygrometer and the pressure bomb show the expected agreement between water potential values with drybeans (Figure 10). The slope was equal to 0.93 with a correlation of R - 0.98. .Calculated whole plant diffusive resistances were approximately the same as the diffusive resistances of individual leaves meas- ured with the parameter (Table 9). However, as the tissue became water stressed and stomates were closing, poor agree- ment between the two techniques was obtained. Sixteen days after transplanting plant water relation parameters were measured. Leaf water potentials of the dis- eased plants were more negative than those of the controls, thus, maintaining a lower leaf water status (Figure 11A). Stomatal resistance to vapor diffusion exhibits a similar pattern since diffusive resistances in the diseased plants were greater (Figure 118) due to stomatal closure. Because the control plants have a higher leaf water potential and less resistance to vapor flaw than do the diseased plants, one can calculate the ease with which liquid water moves through the plant to the leaf surface (Figure 11C). The moderately susceptible cultivar, Swan Valley, responded with a marked decrease in hydraulic conductivity when diseased. NRTER POTENTIRL l-BRRS) 6 099 59 - IKMHUBRKHON CURWES 999 999 999 999 q. 4 4 5 3 3 3 3 ewilted aturgid Figure 9. I T T I I I 60 120 180 240 300 360 TIME (MINUTES) Equilibration curves for the Wescor dewpoint hygrometer at low and high water potentials. '60 O-l a: A 0“ (D CI. c: (g—l a: a-O CD I *‘ U '4 an 33‘ 2 Cd 8 .. m — a: D - 03 :3 .. slope-.93 20.07 SE E - R-.98 N >P=0.001 o I I I I I I I I I 0 2 4 8 B 10 12 14 IS 18 DENPUINT HYGROHETER [-BHRS) Figure 10. Relationship of leaf water potential values between dewpoint hygrometer and the pressure bomb for excised dry bean leaf tissue. Figure 11. 61 Measured and calculated water relation parameters of well-watered dry bean plants. Values are the means of 4 replicates for each treatment. The leaf water potential (A), diffusive resistance (B), hydraulic conductivivy (C) and transpiration rates (D) were all affaected in the moderately suscep- tible cultivar, Swan Valley, but had little effect on the moderately resistant cultivar NEP-Z. 62 cuf o o v N a 9 , n N _ 153%. 32228,. mimics are x $35 Rs. 282.52? o— .o o v a o 12%-. $225.: $2: ham: 9...: x a. o m\¢