ABSTRACT EFFECTS OF NURSERY SOIL FUMIGATION ON GROWTH AND PHOSPHORUS NUTRITION OF PINE AND SPRUCE SEEDLINGS BY Jean-Paul Campagna In a large scale nursery seedling production soil fumigation is an important and necessary technique to control parasitic organisms. In Quebec nurseries some adverse side effects of fumigation treatments have been observed including what appears to be severe P deficiency. This study seeks to define the influence of forest nurs- ery soil fumigation on the N and P nutrition of spruce and pine. Greenhouse and field studies showed that soil fumigation with vapam (sodium methyl dithiocarbamate) or methyl bromide (bromomethane), or soil heat sterilization (greenhouse). applied without supplemental P was associated with a decrease in growth and a deficiency of P in red pine (Pinus resinosa, Ait.) and white spruce (Picea glauca Jean-Paul Campagna (Moench) Voss) seedlings, while the addition of P (336 or 505 kg P/ha) significantly increased the total bio- mass and the shoot P concentration. Ammonium-N'was a better source of N than NO3-N for the development of conifer seedlings. Soil fumigation, NH4 sulfate and superphosphate appeared to be the best combination for raising red pine and white spruce seedlings. After two growth seasons much larger seedlings were still obtained following soil fumigation with appli- cations of superphosphate and NH sulfate than with any 4 other treatment. Soil fumigation and P addition were related to a significant soil pH increase. Nitrification was hindered by soil fumigation and NH4 accumulated in the soil for a portion of the season. The level of P in soil was signif- icantly increased only after addition of superphosphate or bone meal. Inoculation of vapam or methyl bromide fumigated soil with mycelium from mycorrhizal fungi pure cultures failed. However, red pine and white spruce seedlings grown in the same soil inoculated with forest soil showed a healthy growth and excellent plant development, without the addition of P. NUmerous dichotomously branched short roots were Observed and appeared to be ectotrophic mycorrhizae. Jean-Paul Campagna Hand-made cross-sections of these short roots revealed the presence of intra and intercellular hyphae in an irregular pattern. These are believed to be true, but young ectotrophic mycorrhizae. In the greenhouse, an addition of 6.0 and 9.0 g of forest soil to a fumigated nursery soil did not significantly alter the seedling biomass when compared to the 3.0 g addition. Furthermore there was not a significant interaction of forest soil and P additions on the total biomass of red pine and white spruce seed- lings. In the same experiment, application of 224 to 896 kg P/ha significantly increased the total biomass and shoot P concentration of red pine and white spruce seedlings. The increase may be best characterized by a logarithmic equation of form y = a + b log x. Seed- lings grown in pots without any supplemental P showed P deficiency symptoms and revealed a deficient P level in their shoot tissue at analysis. In nursery seedbeds, application of 336 to 1680 kg P/ha significantly enhanced the growth and the shoot P concentration of red pine seedlings. The seedling growth and shoot P concentration are shown to be Jean-Paul Campagna characterized by a logarithmic equation (y = a + b log x). This curve shape indicates a sharp increase due to the first P additions. An application of 672 kg P/ha to fumigated nursery soil appeared to be an optimum level of P fertilization beyond which there was no significant response. At this level, the seedlings were healthy, large and well balanced. EFFECTS OF NURSERY SOIL FUMIGATION ON GROWTH AND PHOSPHORUS NUTRITION OF PINE AND SPRUCE SEEDLINGS BY Jean-Paul Campagna A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Forestry 1972 ACKNOWLE DGMENTS The author is indebted to the Chairman of his Guidance Committee, Dr D. P. White, for his sustained encouragement and guidance throughout the course of this study. He is also grate- ful to the other members of the Guidance Committee -— Drs J. W. Hanover, S. K. Ries, and A. R. Wolcott -- for their valuable assistance and suggestions during the course of this work. The author extends his appreciation to Drs C. E. Cress and J. M. Tiedje for their guidance and assistance with the statistical aspects and microbiological techniques. Acknowledgment is made to the Department of Lands and Forests, Quebec, for their financial support and their con- tinued interest in this study. A very sincere appreciation is finally expressed to my Wife, Margot, and my son, Michel, for their patience and sacrifice throughout these studies. ii VITA Jean-Paul Campagna Candidate for the Degree of Doctor of Philosophy Final Examination: December 8, 1971. Guidance Committee: J. W. Hanover, S. K. Ries, A. R. wolcott, and D. P. White (Major Professor). Outline of Studies: Major subjects: Soil Science Minor subjects: Herbicides, Plant Nutrition. Biographical Items: Born April 28, 1936, St-Frangois, Co. Montmagny, Quebec, CANADA. Home town: Berthierville, Quebec, CANADA. undergraduate Studies: Laval University, 1956 - 1960 Bachelor in Forestry Science, 1960 Graduate Studies: Laval university, 1960 - 1962 M. S. Forestry, 1962 Michigan State UhiVersity, 1967 - 1972 Ph. D. Forestry, 1972 Experience: Department of Lands and Forest, Berthierville Forest Tree Nursery,Quebec, 1960 to date. Since 1963, Director of the Nursery. Member: Canadian Institute of Forestry Corporation of Forestry Engineers of Quebec Xi Sigma Pi Weed Science Society of America. iii LIST OF LIST OF CHAPTER II III IV TABLE OF CONTENTS TABLES. . . . FIGURES INTRODUCTION. . . . . . . . . . . . . . LITERATURE REVIEW . . . . . . Background. . . . . . . . . . . . . . . Fumigants . . . . . . . . . . Effect of Fumigants on Microbial Populations . . . . . . . . . . . . . Effect of Fumigants on Seedling Growth. Morphological Characteristics and Formation of Mycorrhizae. . . . Role of Mycorrhizal Development in Tree Nutrition . . . . . . . . METHODS OF INVESTIGATION. . . . . Greenhouse (Experiment 1) . . . . . . . Field (Experiment 2). . . . . . . . . . Growth Chamber (Experiment 3) . . . . . Greenhouse (Experiment 4) . . . . . . . Field (Experiment 5). . . . . . . . , . RESULTS AND DISCUSSION. . . . . . . . . A- NITROGEN AND PHOSPHORUS FERTILIZATION OF FUMIGATED OR HEAT STERILIZED NURSERY SOIL - GREENHOUSE (1) AND FIELD (2) EXPERIMENTS. . . . . . . . . . . iv Page vii xi 15 19 24 24 28 30 33 36 39 4O CHAPTER Page 1- Morphological Characteristics . . . . 40 Greenhouse. . . . . . . . . . . . . . 40 Field . . . . . . . . . . 44 First sampling date (14 weeks) . . 46 Second sampling date (I7months). . 50 Discussion. . . . . . . . . . . . . . 55 2- Mineral Nutrient Concentration of Shoot . . . . . . . . . . . . . . . . 57 Greenhouse. . . . . . . . . . . . . . 57 Field . . . . . . . . . . . . . . . . 63 Discussion. . . . . . . . . . . . . . 68 3— Soil Fumigation, Phosphorus Addition and Seedling Survival . . . . . . . . 72 3- Effects of Soil Fumigation on Soil Characteristics . . . . . . . . . . . 74 B- INOCULATION OF METHYL BROMIDE OR VAPAM FUMIGATED NURSERY SOIL - GROWTH CHAMBER (EXPERIMENT 3) . . . . . . . . . 77 Mycorrhizal Formation. . . . . . . . . . 82 Shoot Phosphorus Concentration in Inoculated Pots. . . . . . . . . . . . . 88 c— SUPERPHOSPHATE AND FOREST SOIL INOCULUM ADDITION TO VAPAM FUMIGATED NURSERY SOIL — GREENHOUSE (EXPERIMENT 4) . . . . . . . . . . . . . 90 Phosphorus Concentration in Shoot Tissue . . . . . . . . . . . . . . . . . 95 CHAPTER Page D- SUPERPHOSPHATE ADDITIONS TO METHYL BROMIDE FUMIGATED SOIL - BERTHIER- VILLE NURSERY (EXPERIMENT 5) . . . . . . 98 Morphological Characteristics. . . . . . 99 Shoot Phosphorus Concentration . . . . . 102 V CONCLUSIONS AND IMPLICATIONS FOR MANAGEMENT 107 Nursery Culture Implications. . . . . . . . 108 LITERATURE CITED. . . . . . . . . . . . . . . . . . 112 APPENDIX. . . . . . . . . . . . . . . . . . . . . . 119 vi TABLE LIST OF TABLES TEXT Nitrogen and phosphorus treatments used in Experiment 1 . . . . . . . . Mycorrhizal fungi inoculum used in growth chamber experiment (Experiment 3). . . . . Phosphorus and forest soil inoculum used in Experiment 4. . . . . . . . Rates of phosphorus applied to nursery soil (Experiment 5) . . . . . . . Effect of soil sterilization and fertilizer treatments on morphological characteristics of 16 weeks old red pine seedlings (Experiment 1) . . . Effect of soil sterilization and fertilizer treatments on morphological characteristics of 16 weeks old white spruce seedlings (Experiment 1) . . . Effect of soil fumigation and fertilizer treatments on morphological characteristics of 14 weeks Old red pine seedlings (Experiment 2) . . . Effect of soil fumigation and fertilizer treatments on morphological characteristics of 14 weeks old white spruce seedlings (Experiment 2) . . vii Page 26 31 35 37 41 42 47 48 TABLE Page 9. Influence of experimental factors on morphological characteristics of white spruce seedlings after two growth seasons (Experiment 2). . . . . . . . . . 53 10. Effect of soil sterilization and fertilizer treatments on shoot nitrogen and phosphorus concentration (%) of 16 weeks old red pine seedlings (Experiment 1). . . . . . . . . . . . . . 58 11. Effect of soil sterilization and fertilizer treatments on shoot nitrogen and phosphorus concentration (%) of 16 weeks Old white spruce seedlings (Experiment 1). . . . . . . . . . . . . . 59 12. Effect of soil fumigation and fertilizer treatments on shoot nitrogen, phosphorus and potassium concentration (%) of 14 weeks old red pine seedlings (Experiment 2). . . . . . . . . . . . . . 66 13. Effect of soil fumigation and fertilizer treatments on shoot nitrogen, phosphorus and potassium concentration (%) of 14 weeks old white spruce seedlings (Experiment 2). . . . . . . . . . . . . . 67 14. Effect of experimental factors on first year survival of red pine and white spruce seedlings (Experiment 2) . . . . . 73 15. Influence of experimental factors on pH, NH4 and N03, soil available phosphorus, and exchangeable potassium, calcium, and magnesium . . . . . . . . . . . . . . 75 16. Effect of methyl bromide soil fumigation and mycorrhizal fungi inoculation on morphological characteristics of 16 weeks old red pine and white spruce seedlings (Experiment 3). . . . . . . . . 78 viii TABLE 17. 18. 19. 20. 21. 22. 23. Page Effect of vapam soil fumigation and mycorrhizal fungi inoculation on morphological characteristics of 16 weeks old red pine and white spruce seedlings (Experiment 3). . . . . . . . . 79 Effect of forest soil inoculum and different phosphorus levels on morpho- logical characteristics Of 19 weeks Old red pine and white spruce seedlings grown in a vapam fumigated soil (Experiment 4). . . . . . . . . . . . . . 91 Effect Of soil phosphorus addition on the shoot phosphorus concentration and uptake of red pine and white spruce grown in the greenhouse for 19 weeks; Experiment 4 (meansof 4 replications). . . . . . . . . 96 Effect of soil phosphorus addition on the morphological characteristics, the shoot phosphorus concentration and uptake of red pine seedlings grown in nursery seedbeds for 17 weeks; Experiment 5 (means of 4 replications) . . . . . . . . 100 APPENDIX Preparation of "Hagem" agar for culture of mycorrhizal fungi. . . . . . . . . . . 119 Significance Of experimental factors on morphological characteristics of red pine and white spruce seedlings grown in the greenhouse for 16 weeks (Experiment 1). . . . . . . . . . . . . . 120/121 Significance of experimental factors on morphological characteristics of red pine and white spruce seedlings grown in nursery for 14 weeks (Experiment 2). . 122/123 ix TABLE 24. 25. 26. 27. 28. 29. 30. 31. Significance of experimental factors on morphological characteristics of white spruce seedlings after two growth seasons (Experiment 2) . . . . . . Significance of experimental factors on shoot nitrogen and phosphorus con- centration (%) of 16 weeks old red pine and white spruce seedlings (Experiment 1). . . . . . . . . . . . . . Significance of experimental factors on shoot mineral nutrient concentration (%) of 14 weeks old red pine and white spruce seedlings (Experiment 2). . . . . . . . . Influence of experimental factors on pH, NH4 and N03, soil available phosphorus, and exchangeable potassium, calcium, and magnesium in nursery soil (Experiment 2). Significance of experimental factors on morphological characteristics of red pine and white spruce seedlings grown in the growth chamber for 16 weeks (Experiment 3). . . . . . . . . . . . . . Significance of experimental factors on morphological characteristics of 19 weeks old red pine and white spruce seed- lings grown in a vapam fumigated soil (Experiment 4). . . . . . . . . . . . . . Significance of phosphorus addition on the shoot phosphorus concentration and uptake by 19 weeks old red pine and white spruce seedlings grown in a vapam fumigated soil (Experiment 4). . . . . . . . . . . . . . Significance of phosphorus addition to a methyl bromide fumigated nursery soil on morphological characteristics, shoot phosphorus concentration and uptake of 17 weeks old red pine seedlings (Experiment 5). . . . . . . . . . . . . . Page 124/125 126/127 128/129 l3Q/l3l 132 133 134 135 FIGURE LIST OF FIGURES Effect of methyl bromide soil fumigation, N and P addition on 16 weeks old red pine and white spruce seedling development . . . . . . . . . . . . . . Influence of N sources and superphosphate addition on the shoot dry weight of 16 weeks old white spruce and red pine seedlings grown in methyl bromide fumigated soil (Experiment 1) . . . . Effect of N and P addition on the shoot and root growth of red pine and white spruce grown in methyl bromide fumi- gated seedbed - Berthierville nursery Influence of N sources and superphosphate addition on the shoot dry weight of 14 weeks Old white Spruce and red pine seedlings grown in methyl bromide fumi- gated seedbed (Experiment 2). . . . . . Effect of N and P addition on shoot growth of 2-0 white spruce seedlings grown in methyl bromide fumigated seedbed - Berthierville nursery . . . . . . . . Influence of soil fumigation, N and P addition on the shoot dry weight of 2-0 white spruce seedlings (Experiment 2) . Influence of P and N fertilization on the N concentration (%) in shoot tissue of 16 weeks old white spruce seedlings (Experiment 1). . . . . . . . . . . . Influence Of soil sterilization and P sources on P concentration (%) in shoot of 16 weeks Old white spruce seedlings (Experiment 1). . . . . . . . . . . xi Page 43 45 49 51 52 60 62 FIGURE Page 9. Influence of N sources on N concentration (%) in shoot of 14 weeks Old red pine and white spruce seedlings (Experiment 2)....................64 10. Influence of P sources on P concentration (%) in shoot of 14 weeks old red pine and white spruce seedlings (Experiment 2). . . . . . . . . . . . . . . . . . . . 65 11. Effect of forest soil inoculum addition on the growth of red pine and white spruce grown in methyl bromide fumigated soil. . 81 12. Cross-sections of non-mycorrhizal, and mycorrhizal short root from red pine raised in Rhizopogon roseolus inoculated pot . . . . . . . . . . . . . . . . . . . 84 13. Cross-sections of dichotomous short root of red pine . . . . . . . . . . . . . . . 85 14. Abnormal root of red pine seedling grown in pot inoculated with Amanita rubescens. Cross-sections of these malformations . . 87 15. Influence of forest soil addition on the shoot P concentration and uptake of red pine and white spruce seedlings grown in vapam fumigated soil (Experiment 3). . 89 16. Relation between the shoot dry weight of red pine and white spruce seedlings and the amount of P added to a vapam fumigated soil (Experiment 4) . . . . . . 93 17. Effect of P addition on red pine and white spruce seedlings grown in a vapam fumigated greenhouse soil . . . . . . . . 94 18. Relation between the shoot P concentration of red pine and white spruce seedlings and the amount of P added to a vapam fumigated soil (Experiment 4) . . . . . . 97 xii FIGURE 19. 20. 21. Page Relation between the shoot dry weight of 17 weeks Old red pine seedlings and the amount Of P added to a methyl bromide fumigated nursery soil (Experiment 5). . . . . . . . . . . . . . 101 Relation between the shoot P concentration and the P uptake of red pine seedlings and the amount of P added to a methyl bromide fumigated nursery soil (Experiment 5). . . . . . . . . . . . . . 103 Relation between the P concentration in shoot and the shoot weight of 17 weeks old red pine seedlings (Experiment 5) . . 105 xiii CHAPTER I INTRODUCTION The success of tree seedling production in forest nurseries depends largely on how effectively soil-borne dis— eases and parasitic organisms are kept under control. Fumi- gants such as methyl bromide, vapam (sodium methyl dithio- carbamate), and vorlex (mixture of methyl isothiocyanate and chlorinated hydrocarbons) are now effectively used in forest nurseries to control these disease organisms. The fumigants are also very effective in destroying most weed seeds. Therefore these broad spectrum fumigants are a tool of every nurseryman and have gained wide acceptance as dual-purpose soil treatments (White and Potter, 1963). Early forest tree nursery research in chemical damping-Off and weed control dates back to at least the 1920's when Toumey and Korstian (1942) were experimenting with sul- furic acid, copper and zinc sulfates. The use of soil fumi- gants, a relatively new science, has really been developed in the last 15 or 20 years. The fumigants were most primarily used in horticultural and vegetable crops (Kock, 1951), and they were brought into the forest nurseries only in the early 1950's to solve the problem of soil-borne diseases (Howe and Clifford, 1962). Seedling diseases not only reduce germination, but they also affect seedling vigor and consequently their ability to survive other unfavorable environmental conditions. Soil- borne diseases are devastating in a forest tree nursery. Damping—Off, one of the worst, can completely destroy entire seedbeds of young coniferous seedlings. The intensity of this disease varies with the pH of the soil, the weather, method and time Of sowing and the seedling species. Formaldehyde is known as one of the first soil dis- infectants used in forest nurseries. In the early 1950's fumigants of the bromide and methyl bromide type appeared and they are still largely used to-day even if others such as vapam, vorlex, mylone, etc. are also applied in forest nurs- eries against soil-borne diseases and weeds. Their selec- tivity, fungicidal and herbicidal properties make them in- dispensable in large nursery operations. However, in many cases these fumigants do show a residual effect on the growth of young coniferous seedlings. This seems to be related to their action on the balance of microbiological life in soil. Iyer and Wilde (1965), studying the effects of many biocides and fumigants on the nutrient status of seedlings, found that unbalanced or large top/root ratio of conifer seedlings was related to soil treated with vapam. Others such as Henderson and Stone (1970) reported coniferous seedlings deficient in P following a soil fumigation. More- over these seedlings grew poorly and had a red purple dis- coloration of their needles. This poor growth was related to an absence of mycorrhizal development. Meanwhile Howe and Clifford (1962) reported very good growth or a "ferti— lizer effect" after soil fumigation with methyl bromide and attributed it to a reduction in weed competition and a soil free of pathogens. It is now accepted that this decreased growth and purple discoloration of coniferous seedlings represents a deficiency of P in conifer seedlings caused by a decrease in P uptake in the absence of mycorrhizal development. Supplying a very high amount of P to the nursery soil allows adequate P nutrition even without mycorrhizae and results in good growth (Henderson and Stone, 1970). In several Quebec nurseries, it was observed that adverse residual effects from fumigation interfered with the production of healthy seedlings. The objectives of this study were: (1) to examine the possible sources of available phosphorus and suitable application rates which can be used to stimulate the growth of coniferous seedlings after soil fumigation and (2) to assess the ameliorative influence of inoculation with forest soil or pure cultures of mycorrhizal fungi after soil fumi- gation in greenhouse and field conditions. Red pine (Pinus resinosa Ait.) and white spruce (Picea glauca (Moench) Voss) were used as the indicator species. CHAPTER II LITERATURE REVIEW Background The gardener, nurseryman, greenhouse Operator has long been fighting a battle with plant diseases caused by soil-borne microorganisms. These organisms which live in the soil cause a variety of diseases which trouble those who work in this area. It has been extremely difficult in the past to gain control over these microorganisms through the use of cultural practices. The operators of many large greenhouses have sterilized their soil by steam, but this method is costly and not always completely effective. Furthermore this method is hardly performed in nursery seedbeds. Then the nurserymen were at the mercy of the fungi until the development of soil fumigants. The first fumigants used extensively in forest tree nurseries, horticultural and vegetable crops were of bromide or methyl bromide types. For many years methyl bromide has been the standard by which most wide-spectrum soil fumigants have been compared. Methyl bromide is a colorless and nearly odorless liquid or gas. It is applied in this form under a poly- ethylene covering. This biocide is effective in repressing soil-borne diseases and parasites that attack germinating seeds and young seedlings. It is also efficient in con— trolling weeds. Methyl bromide can also be mixed with other fumigants such as chloropicrin, propargyl bromide to give several new compounds: Methyl bromide MC-2 (98% methyl bromide, 2% chloripicrin), Trizone (61% methyl bromide, 30% chloropicrin, and 9% propargyl bromide), and Dowfume MC-33 (67% methyl bromide, 33% chloropicrin). One of the most notable attributes of these compounds is their broad spec- trum of biological activity. (The high biological activity of these fumigants has aroused much interest and they gained wide acceptance amongst nurserymen. Since the early 60's several other products of which vapam is one, have been used. Vapam is a water-soluble liquid containing 32.7% sodium methyl dithiocarbamate. Applied to the soil as a preplanting treatment it is con- verted into a gaseous fumigant (methyl isothiocyanate). Vapam is widely used for the control of weeds and soil-borne pests that attack ornamental, food and fibre crops, and to- bacco seedlings. Waksman (1966) stated, "The soil is not a mass of rocks and residues; it is not a dead organic-inorganic sys- tem, but a living system teeming with numerous forms of life". Of the several forms of life mentioned roots of higher plants and certain fungi are involved in intriguing natural phenomena. Specific fungi grow upon and vigorously invade portions of root systems that are primarily respon— sible for nutrient absorption by higher plants. The term "mycorrhyza" meaning fungus—root, designates these partic- ular invasions of roots by fungi. Without mycorrhizae, many plants including our most important timber species, could not survive in the dynamic, fiercely competitive bio- logical communities that abound in natural soil habitats. In nurseries, the addition of any potentially toxic mole- cule constitutes a serious threat to the presence of mycor- rhizal fungi and their association with seedling roots. Fumigants Fumigants can be applied to the soil as a liquid or in gaseous form. As a gaseous chemical they are applied under a covering at the soil surface and move into the soil. If the chemical is in a liquid form it is injected into the soil at a depth of 4 to 6 inches, and covered. The injected fumigant evaporates and diffuses throughout the soil. To be effective a fumigant must dissolve in soil water, because it is only in this form that it can kill nematodes, fungi and weed seeds. If the soil is dry, the fumigant is not absorbed or dissolved in water and it diffuses rapidly throughout the soil. Then an Optimum con- centration of the fumigant is not retained in the soil mois- ture resulting in little effect. Under moist conditions, the diffusion is not so rapid, but the greatest part of the fumigant is in the soil moisture where it is needed (Howe, 1965; White, 1965). The ideal type of soil for fumigants is a sand or a sandy loam which permits a much better diffusion and distribution of fumigants than clay soils. If soil temperature is too low (below 4.50 C), it stops the evaporation of the fumigants and thus their diffu- sion. The most common temperature of application of fumi- gants is about 160 C, with a range of 100 C to 300 C. Other factors in the effectiveness of fumigants are the length of exposure, the amount applied and the covering. Methyl bromide must be applied under a tarp set up before the application in surface of the soil, or injected into the soil which is covered as soon as possible after application. Vapam can be injected into a soil which has to be sealed with water or covered with a tarp, or it can also be drenched into the soil. When only drenched into the soil vapam is not so effective. In forest nurseries, fumigants have principally been used until now in a three-way action. Indeed they are used to kill fungi and control most of the common soil-borne plant parasitic diseases such as damping-off and root rot of seedlings. They are also utilised in order to control most of the usual soil-borne plant parasitic species Of nematodes causing severe damages to root seedlings. Furthermore they are used to kill weed seeds (Delong, 1960; Harrison, 1966). Effect of Fumigants on Microbial Populations The soil is a very complex and dynamic system; its microscopic inhabitants such as fungi, bacteria and actino- mycetes are essential to plant growth and to soil fertility. By their enzymes they can decompose roots and crop residues and make nitrogen available to plants. They also form NO3 from NH4 fertilizers and transform P to compounds assimilable by plants. Moreover the soil microorganisms perform many other processes useful or harmful to plant (Alexander, 1958). The soil fertility often depends on the delicate balance existing between these various types of microor- ganisms whose activities determine the efficiencies of the carbon, nitrogen, mineral, etc. cycles which they regulate. It is obvious that the addition of any potentially toxic molecule constitutes a serious threat to this equilibrium and to its fertility. The methyl bromide fumigants control fungi such as Rhizoctonia solani Kuchn, Phytophtora spp. and Pithyum spp 10 which cause damping-off and heavy losses of forest tree seed- lings at emergence. They also decrease considerably the mor- tality due to root rot disease by killing the fungus Macro- phomina phaseoli (Maubl) (Smith and Bega, 1966; Turner, 1960). Fumigants also have an effect on fungi related to mycorrhizal infections of tree roots. It seems that fumi- gants at least kill the vegetative stage of these fungi in soil; indeed quite a few authors reported that conifer seed- lings growing in fumigated soil do not show any mycorrhizal infection the first year of growth (Howe and Clifford, 1962). Iyer and Wilde (1965) reported that a vapam treatment to a nursery soil in Wisconsin nearly eliminated mycorrhizal short roots and fibrous laterals with resulting 75 percent decrease in the absorbing capacity (titration value) of red pine roots. The same phenomenon was observed on Ponderosa pine and Douglas fir (Wright, 1964). After a second year of growth, roots of seedlings from fumigated beds are most of the time comparable in mycorrhizal development to those from unfumigated beds. Then it appears that mycorrhizae, even though drastically reduced by soil fumigation, regen- erate rather quickly and within two years reach levels comparable to those in untreated plots. The absence of mycorrhizal development is most of the time related to a 11 red purple discoloration and a poor develOpment of the seed- lings. This effect can be alleviated by a high amount of P in the soil solution (Henderson and Stone, 1970). The bacteria, an important group of soil flora, are essential for the maintenance of its fertility. Wensley (1953) studied the effect of soil fumigation with Dowfume MC-2 on 4 physiological groups of bacteria. He noted that the nitrifiers (Nitrosomonas, Nitrobacter) and certain cellu- lose decomposing bacteria are more susceptible to methyl bromide than the denitrifier or ammonifier bacteria. While nitrification is suppressed by methyl bromide, ammonifica- tion is slightly reduced. The resulting lag in nitrification may last eight to ten weeks or more (Good and Carter, 1965). Hence fumigation produced an unbalanced ratio in NO3-N rela- tive to that of ammonia N in the soil. But this fact seems to be slightly favorable to conifer seedlings which seem to prefer NH4 to NO as N source (McFee and Stone, 1968). 3 In general microbial numbers are initially decreased by fumigation, but most of the time many organisms will quickly reinvade the soil, including beneficial fungi and soil bacteria. Sometimes their numbers may exceed those in untreated soils. Several factors may contribute to this fact. The cell material of the organisms killed may offer 12 a ready source of energy and carbon material for the living organisms. But the most important factor seems to be the reduced competition. Usually Trichoderma viride is the first fungus species to reinhabit fumigated soils. It often survives fungicide treatment and quickly develops in an environment which is less competitive. If annihilated by the treatment, it may reinvade the soil in which a sufficient amount of the chemical is still present to prevent the development of other species. Since there is no competition, it may grow faster than other organisms and become dominant. "The changes in the microbial population of the soil following treatment with fumigants may exert a biological control effect on root parasites. Trichoderma viride, which most commonly becomes dominant following partial soil steriliza- tion with chemicals, is a well known antagonistic species. It has been shown to exert an antagonistic influence on Phytophtora, Pythium, Armillaria, Rhizoctonia, and other parasitic forms" (Martin and Pratt, 1958). Effect of Fumigants on Seedling Growth The effects of microbiological decomposition, chem- ical absorption, adsorption on soil colloids and losses by leaching upon the breakdown of these fumigants in the soil are not fully understood. However it is generally agreed 13 that the major disappearance is by volatility. Indeed we must allow a period of aeration before sowing in order to permit the volatile portion of these fumigants to dissipate into the air. The breakdown of methyl bromide fumigants involved a lag period in the build up of responsive organisms, and an induction period for adaptive enzymes to metabolize the chem— icals (Hollis, 1964). Soils fumigated with these chemicals hold residues containing bromide ions which are troublesome to a few species of plants (Worsham, 1964). A soil treated with Trizone shows an increase in soluble chlorides which may be harmful to plants if they are planted too soon following fumigation (Wright, 1964). Soluble residues, such as those cited above, are reported capable of causing plant damage often located at the root level. Red and white pine 1-0 seedlings show lit- tle or no mycorrhizae when growing in a soil treated with Dowfume MC-Z or Trizone while untreated seedlings show numerous mycorrhizae. However at 2-0 there is no differ- ence in roots of seedlings from treated or untreated seed- beds (Howe and Clifford, 1962). Therefore it is obvious that the mycorrhizae fungi have reinvaded the treated area. Wright (1964) also reported that Trizone significantly de- creases the mycorrhizal formation during the first growth year of Ponderosa pine and Douglas fir. 14 For several years, at the Berthierville nursery, Quebec, severely stunted 1-0 pine and spruce seedlings in spots throughout the seedbeds have been Observed following an application of fumigants. The stunted seedlings also showed a reddish purple coloration resembling P deficiency, and a lack of mycorrhizal development. Henderson and Stone (1970) observed that both growth and P content of non- mycorrhizal coniferous seedlings grown in fumigated soil without P fertilization were less than any other treatments: they attributed this phenomenon to the absence of mycorrhizae. In these New York experiments, inoculation with either mycor- rhizal roots or untreated soil, or treatment with high appli— cation of inorganic P fertilizer, corrected the reduced P up- take and poor growth caused by fumigation alone. Iyer, Chesters and Wilde (1968) in Wisconsin report: "Some of the recently introduced organic biocides abnormally stimulate the growth of crowns of nursery stock, but depress the develop— ment of root systems. In consequence, reforestation material exhibits an extreme degree of succulence, low specific grav- ity abnormally high top: root ratio, greatly reduced titra— tion value of roots, and impeded development of mycorrhizae. The latter deficiency hinders the uptake of P even when this element is abundant in the soil in available form". It seems that, following the experiments of these authors, 15 nursery plants lose their capacity for a normal uptake of P when we have either a growth stimulation or reduction following a soil fumigation. As these coniferous seed- lings are characterized by an absence of mycorrhizal root development, this helps to point out the role of my- corrhizae in normal P and nutrient uptake which leads to production of balanced seedlings. Morphological Characteristics and Formation of Mycorrhizae On the basis of interrelation between the fungus hyphae and the root cells, mycorrhizae are classed in two main groups, ectotrophic and endotrophic. The kind is usually specific for the genus of higher plant in which it occurs, and mycorrhizae have been found on most genera of seed plants that have been examined. Ectotrophic mycor- rhizae are common on the pine (Pinaceae) family among gym- nosperms and on the birch (Betulaceae), beech and oak (Fagaceae) and a few other families among angiosperms. Endotrophic mycorrhizae are present on the roots of most shrubs and certain trees as maple, yellow poplar, sweet gum, redwood and apple among others (Hacskaylo, 1967). Typical ectotrophic mycorrhizae are caused by in- vasion of actively growing absorbing roots usually by hymenomycetous, but sometimes by ascomycetous, fungi 16 (Hacskaylo, 1957). Mycorrhizal association is usually con- fined to those roots in the top few inches of soil. The smallest of the secondary roots are invaded by fungi dur- ing periods of active growth. With few exceptions, the fungi involved (nearly all basidiomycetes) produce mush- rooms as fruiting bodies. Attachment Of hyphae to tree roots apparently is requisite to fruiting under natural conditions. Even to-day the principles governing the entrance of the hyphae of the fungal symbiont into the roots are far from clear and we can synthesize only a partial explanation of the mechanisms of the association. First there is a contact between actively growing roots and compatible fungi. The contact may come from spores germinating in the vicinity of the roots or by ex- tension through the soil of hyphae from either residual mycelium or established mycorrhizae. Bjorkman (1942) states that the mycorrhizal fungi seeking soluble carbohydrates enter the roots only if a surplus of such sugars is present in the roots. According to this theory a surplus of soluble sugars becomes a main factor governing the initiation and the establishment of the symbiotic relationship. Meanwhile growth of mycor- rhizal fungi on the surface of the roots is greatly l7 stimulated by exudates from the roots. These exudates con- tain at least one growth promoting metabolite that Melin (1954) designated as "M" factor. This substance has not been identified and the dependency on the "M" factor varies with the species of mycorrhizal fungi. Entrance of ectotrophic mycorrhizal fungi into the roots requires secretion of pectolytic enzymes, which dis- solve the middle 1ame11ae and thus permit the hyphae to grow through the intercellular region of the cortex. The pattern formed by hyphae in the cortex is called a "Hartig net". The meristematic tips and the vascular cylinder are not invaded. The fungus and root cells retain their vital characteristics and show no pathological symptoms (Hacskaylo, 1957). The invading organisms in endotrophic mycorrhizae are primarily inconspicuous phycomycetous fungi that produce subterranean, nearly microscopic fruiting bodies (Hacskaylo, 1967). Endotrophic fungi are present on surface of the my- corrhizal rootlets as individual threads or loose hyphae wefts. The fungi secrete cellulolytic enzymes that dis- solve a minute portion of the cell wall. This allows the hyphae to penetrate root hairs and other epidermal cells. Beside the two types of mycorrhizae described, there occasionally appears on tree roots, primarily in 18 nurseries, the typical intercellular organization of the ectotrophic mycorrhizae plus intracellular penetration by hyphae. These ectendotrOphic mycorrhizae are sometimes thought to represent a transitional stage between the ecto- trOphic and the endotrophic type. Conversion of roots into mycorrhizae is accompanied by considerable changes in the physiology of these roots. Generally these physiological changes are demonstrated by renewed meristematic activity in Old short roots, swellings caused by growth of new cortical cells, sometimes dicho- tomous branching of roots, and inhibition of root hair de- velOpment on the swollen parts (Slankis, 1961). Then it seems logical to presume that a profound change in the physiology of these roots is a prerequisite for the estab- lishment of the symbiotic relationship. Several factors are considered important for a normal mycorrhizal root development of trees. Mycorrhizae of trees develop most extensively in acidic soils; indeed mycorrhizal fungi of trees studied for pH requirements are acidophilic. These fungi also require certain vitamins and amino acids, available in sufficient amounts in the soil for maximum growth of the fungi. Glucose and other sugars seem to be the main source of carbon of these fungi (Melin, 1953). 19 Abundance of new mycorrhyzae is also correlated with higher soil moisture levels during spring and autumn as com— pared with those in summer. The soil should be well aerated for good development of mycorrhizae. Low light intensities are a limiting factor in the formation of mycorrhizae on both natural and long days (Hacskaylo and Snow, 1959). The results of several investigators consistently show that formation of ectotrophic mycorrhizae varies in- versely with soil fertility. Fowells and Krauss (1959) con- firmed the findings of Hatch (1937) that mycorrhizae are generally more abundant with low levels of N and P. Exper- iments of Hacskaylo and Snow (1959) further support these conclusions. Daft and Nicholson (1966), who studied three Endogone endophytes on plants grown in sand and watered with a nutrient solution, obtained the largest increases in growth of mycorrhizal plants under conditions of low P avail- ability. Role of Mygorrhizal Development in Tree Nutrition The significance of mycorrhizal fungi as a persis- tent component of the forest soil microflora and the impor- tance of these fungi in tree growth can no longer be denied. In 1917 Professor Elias Melin, at Uppsala, Sweden, initiated new-and significant approaches to studies on mycorrhizae of pine, spruce and other Scandinavian trees. He observed that 20 in drained peat bogs those trees whose roots become mycor- rhizal grew normally. Thereafter he directed his efforts toward research on the physiology of tree mycorrhizae under carefully controlled laboratory conditions. At that time he hypothesized that organic N compounds might be absorbed by the hyphae and translocated into the root tissues. Hatch (1937), by analyzing tissues of mycorrhizal versus non-mycorrhizal seedlings, found increases of 86 percent N, 234 percent P, and 75 percent K over the non- mycorrhizal seedlings. He theorized that mycorrhizae were considerably more effective in nutrient uptake than non- mycorrhizal roots because of the increased surface area provided through root proliferation and of large absorbing surfaces of hyphae in the mantle and surrounding soil. His data showed that mycorrhizae were most effective in soil moderately deficient in N, P and K. Other worker findings supported these theories. Kramer and Wilbur (1949) observed that mycorrhizae of pine accumulated radio—active P to a greater degree than non-mycorrhizal pine roots. Melin and Nilsson (1957) clearly demon- strated that mycorrhizal fungi transported radio-active inorganic and organic N, P, Ca, and Na from the substrate into roots of Scotch pine. The efficiency of the 21 mycorrhizal system was far greater than in the non- mycorrhizal root. Harley (1959) indicated that there is a certain difference in metabolism between mycorrhizal and non—mycorrhizal roots. Gray and Gerdemann (1967) also demonstrated that mycorrhizal sweetgum (Liquidambar styraciflua) and tulip- tree (Liriodendron tulipifera) accumulated larger quantities of P32 than did non-mycorrhizal plants. The use of P32 brings evidence that ectotrophic mycorrhizal fungi play a very active role in nutrient uptake by trees and the en- hanced ability of mycorrhizal plants to absorb P. Baylis (1967) concluded that, in New Zealand forest soils which are normally low in available P, mycorrhizae are essential for the uptake of enough P to bring about normal growth. Soils in many parts of the world have been found devoid of fungi that form ectotrophic mycorrhizae. At- temps to establish trees that normally possess ectotrophic mycorrhizae in some of those areas have failed several times. Jorgensen and Shoulders (1967) reported that the presence of visible mycorrhizae on roots of seedlings of slash pine (Pinus elliottii Engelm) had a significant and important beneficial effect on the survival of the seed- lings, when planted on deep sandy soils in northwest Louisiana and on sandy loams in the central part of the 22 United States. Wilde (1968) reported that in prairie and other grassland soils devoid of certain symbiotic fungi, the growth of trees is arrested at the stage of the first whorl of leaves; during the first growing season in such soils they exhibit symptoms of malnutrition and die. An introduction of fungal symbionts of trees into grassland soils, accomplished by an addition of a fraction of 1% of a forest soil or a few crushed roots of mycorrhizal seed- lings invariably leads to a rapid initiation of a vigorous growth of tree seedlings. Mycorrhizae are not only generally recognized to aid tree growth and be necessary for tree survival on many sites, but Zak (1964) suggested that ectotrophic mycor- rhizal roots may be less susceptible than non-mycorrhizal roots to infection by root pathogens. He postulated that mycorrhizal fungi may protect absorbing roots of trees by: (i) utilizing root carbohydrates and other chemicals, thereby reducing the attractiveness of the root to patho- gens; (ii) providing a mechanical barrier to the patho- gens in the form of a fungus mantle; (iii) secreting anti- Iliotics which may inhibit or kill potential pathogens: and (ill) attracting, while in mycorrhizal association with the hOSt root, a protective rhizosphere population of other m ‘1 Q roorganisms . 23 Mycorrhizal fungi may afford protection also by stimulating host root cells to elaborate inhibitors that may maintain the symbiotic state and that also serve to inhibit infection by pathogens. Marx and Davey (1969a, 1969b) showed that ecto- trophic mycorrhizae formed aseptically by several symbiotic fungi on the roots of shortleaf (Pinus echinata Mill.) and loblolly (P. taeda L.) pine seedlings were resistant to in- fection by zoospores of Phytophthora cinnamoni Rands. The findings confirmed Zak's (1964) theory that ectotrophic my- corrhizal fungi function as biological controls against pathogenic root infections. It is apparent that the use of biocides in nurs— eries can reduce the mycorrhizal development of tree seed- lings. This effect should not be overlooked and the damages caused to the absorptive systems of the seedlings should not exceed the initial benefits from control of disease and reduction of competition. Indeed ectotrophic mycorrhizae seem to function not only as physiological entities bene- ficial to plant health, as reported by many researchers, but also as biological controls against pathogenic root infections. CHAPTER III METHODS OF INVESTIGATIONS Greenhouse (Experiment 1) The influence of soil sterilization (fumigation or autoclaving) and different N and P sources on the growth Of red pine and white spruce seedlings was measured in this first greenhouse study. The seedlings were grown in 500 ml plastic containers containing 0.45 kg of sandy loam nurs— ery soil from the Berthierville nursery.l A sample of this soil was analyzed for NO3-N by the Brucine method and NH4-N by the Nessler reagent method (Greweling and Peech, 1965). Available P was determined by the Bray Pl technique. Ex- changeable cations were determined in ammonium acetate ex- tracts: K by flame photometer, Ca and Mg by atomic absorption. Soil reaction was read with a pH meter using a 121 soil water ratio and organic matter was determined by loss on ignition at 5000 C. Soil analyses by the Michigan State university Soil Testing Laboratory showed the following soil characteristics: lQuebec Provincial Nursery. 24 25 Organic Available Nutrients (ppm) pH Matter (%) NH4-N NO3-N P K Ca Mg 5.8 4.2 5.0 4.0 81 73 840 18 The screened soil (6.2 mm mesh) was first moistened, then divided in three parts. A part was autoclaved at 1210 C and a pressure Of 1.06 kg/cm2 for two and a half hours. A second part was fumigated with methyl bromide (bromomethane). The pots (9.45 kg of soil) were put in large polyethylene bags and the fumigant was injected in the bags at a rate of 0.9 kg MB/9.3 square meters of soil surface. After two days at room temperature (220 C) the bags were Opened to permit aeration for three days. The third part of soil was kept as control. Fertilizer treat- ments were applied after the autoclaving, or before the fumigation (Table 1). In each pot we mixed with the soil potassium sulfate at a rate of 90 kg K/ha. Phosphorus (336 kg P/ha) as superphosphate was applied as a band 2.5 cm below the soil surface. On April 13, half rate (rate: 112 kg N/ha) of N sources were applied and a month later we applied the second half of the treatment. Red pine and white spruce seeds stratified in moist medium for a month were sown and pots placed on a greenhouse 26 Table l. Nitrogen and phosphorus treatmentsl used in experiment 1. FERTILIZER TREATMENT N P O 0 Control 0 Rock Rock Phosphate (8.8% P) 0 Super Superphosphate (20% P) 0 Bone Bone Meal (4.8% P) N03 0 Sodium nitrate (16% N) NO3 Rock Sodium nitrate + rock phosphate NO3 Super Sodium nitrate + superphosphate NO3 Bone Sodium nitrate + Bone meal NH4 0 Ammonium sulfate (20% N) NH4 Rock Ammonium sulfate + rock phosphate NH4 Super Ammonium sulfate + superphosphate NH4 Bone Ammonium sulfate + Bone meal lNitrogen at 112 kg of N/hectare of P/hectare: equivalent to 100 P/acre. and phosphorus at 336 kg lbs N/acre and 300 lbs 27 bench on March 10, 1968. The pots were periodically watered with distilled water to adjust soil moisture to approximately 20 per cent by weight as determined by weighing the pots. Several pairs of fluorescent lights were arranged parallel in the center of the plant bed 64.0 cm from the plant foliage level. These lights were auto- matically controlled and synchronized with the day photo- period to provide artificial light for 18 hours per day. The experiment was arranged in a randomized complete block design with 4 replications. Sixteen weeks after sowing, measurements were made of seedling length from root collar to terminal bud, dry weight of both shoots and roots. We also made observations on the mycorrhizal development of the root system. The plants were dried at 700 C for 48 hours in a mechanical convection oven, ground in a Wiley mill. Total N was determined on shoots by the micro-Kjeldahl method. Phosphorus was measured using photoelectric spectrometer at the MSU Horticulture Laboratory. The data were subjected to analysis of variance and treatment means were compared using planned orthogonal contrast and Tukey's w-procedure. 28 Fieldijxperiment 2) In this experiment we wanted to test the interaction of vapam (sodium methyl dithiocarbamate) and methyl bromide fumigants with adjustment of N and P fertility in red pine and white spruce seedbeds at the Berthierville nursery in Quebec in the Spring of 1968. The fertility of the sandy loam nursery soil is outlined below: Organic Available nutrients (ppm) pH Matter (%) NH 4-N NO3-N P K Ca Mg 5.2 3.8 8.0 4.0 120 290 740 45 A split-plot design with a factorial arrangement of treatments was used. Fumigant treatments were the main units, whereas fertilizer treatments (Table l) were the sub- units. The P fertilization (505 kg of P/ha) was applied on May 11, but N (112 kg of N/ha) application was delayed until July 2 after seedling germination. Methyl bromide (0.9 kg per 9.3 square meters) and vapam (600 cubic cm per 9.3 square meters) were applied to the soil on May 13. Non-fumigated seedbeds were used as a control. Stratified seeds were sown on May 30-31. 29 Total precipitation at the field plot site for the 1968 growing season was inferior as compared to the eight precedent years. Spring and early summer precipitation was nearly normal but July, August and September were below normal and we supplied by overhead irrigation. Total pre— cipitation for the 1969 growing season was a little above normal as compared to the 1959 to 1967 seasons. Above normal precipitations were recorded in April, May, June and July, but below normal in August. A comparison be- tween the experimental seasons 1968 and 1969 and the period of record is shown below: Growing Season Precipitation (cm) April May June July August Sept. Total Period of Record 6.3 5.7 7.5 9.2 10.3 7.7 46.7 (1959 - 1967) 1968 Growing 4.8 7.6 7.1 5.5 4.4 3.3 32.7 Season 1969 Growing 9.4 11.7 8.8 12.1 5.8 7.4 55.2 Season At the end of August, the seedlings were counted and, on September 4, 25 seedlings were collected in every sub-plot. The physical growth measurements, plant chemical analyses, and tests for significance among treatment means 30 were the same as described previously. A complete soil sampling was also made and soil samples were subsequently analyzed. At the end of the second growth season (October 69) 10 white spruce seedlings were collected in every sub-plot and their total length, their dry weights of shoots and roots were measured. Growth Chamber (Experiment 3) Since it was apparent that mycorrhizal fungi are killed by fumigation we wanted to inoculate our soil in order to give it the microorganisms necessary to a good mycorrhizal development of seedling roots. To inoculate the fumigated soil, forest soil and pure cultures of mycorrhizal fungi were used. The soil inoculum was a soil from a red pine plantation located at Kellogg Experimental Forest near Battle Creek, Michigan. The isolates of mycorrhizal fungi were kindly furnished by Dr V. Slankis1 and Dr E. Hacskaylo.2 The isolates were first cultivated on "Hagem" agar in petri dishes lDr Vladislavis Slankis, Department of Fishery and Forestry, Canadian Forestry Service, Maple, Ontario. 2Dr Edward Hacskaylo, Plant Physiologist, Forest Physiology Laboratory, Beltsville, Maryland 20705. (Appendix Table 21). Chunks of mycelium were used to in- oculate fumigated soil with mycorrhizal fungi (Table 2). Table 2. Mycorrhizal fungi inoculum used in growth chamber (Experiment 3). SEEDLING SPECIES INOCULUM SOURCE Red Pine White Spruce Nil Forest soil Suillus luteus Amanita rubescens Rhizgpogon roseolus Nil Forest soil Suilluslgganulatus Cenococcum graniforme Telephora terristris Soil collected in August 68 at Berthierville nurs- ery was dried and screened using a 6.2 mm mesh. The soil characteristics are outlined below: Organic 4— Available Nutrients (ppm) PH Matter (%) NH NO3-N P K Ca Mg 5.2 3.8 6.5 5.5 70 130 450 24 32 The soil was first moistened and then put in 4.4 liter plastic containers containing 2500 g soil and 1200 g quartz sand. These pots were fumigated with methyl bro- mide (0.9 kg/9.3 square meters) or vapam (840 liters/ha). Pots were kept in polyethylene bags during three days and then aerated for four days before sowing. Red pine and white spruce seeds were sterilized with 30% hydrogen peroxide for one minute, washed five times with sterile water and put on water agar to germinate. At sowing time chunks of mycelium were buried in the soil at a depth Of 3 to 4 cm and germinated seeds (radicles 0.2 - 0.5 cm long) were put in the soil. The soil inoculum (5 gm) was mixed in the 4 top cm of soil. The environmental conditions maintained throughout this experiment were a 18 hour light period at 26.60 C, a 6 hour dark period at 160 C, and a relative humidity of approximately 65 per cent. The light intensity was approx- imately 2600 foot candles at the plant foliage level. The pots were periodically watered with sterile distilled water to adjust soil moisture to approximately 20 per cent by weight as determined by weighing the pots. The experiment was arranged in a randomized com- plete block design with 4 replications. After 16 weeks, the number of mycorrhizal and unmycorrhizal short roots 33 was counted using a 10X magnification, and also the number and the length of long roots were recorded (4 seedlings per pot). Numerous root sections were fixed in formaldehyde- acetic acid—alcohol and sections were stained and examined under greater magnification to be sure of the presence or absence of mycorrhizae. Green and dry weight measurements were made of seedling shoots and roots. The total length of the seedlings was also recorded. Moreover the P con- centration of seedling shoot tissue was determined for a few samples. The data were subjected to analysis of var— iances as described previously. Greenhouse4(Experiment 4) In the previous experiments superphosphate or forest soil inoculum addition to fumigated nursery soil brought a better seedling growth than any other treatments. In this experiment these two treatments are put together to check their possible interaction on conifer seedling development. A soil similar to experiment 3 was used. Screened soil (2000 g) was placed in 3.7 liter plastic greenhouse containers and fumigated with vapam at a rate of 840 li- ters per hectare. As in the previous experiment pots were placed in sealed polyethylene bags for 3 days and then aerated for 6 days before sowing. 34 The P fertilizer (Triple superphosphate, 20% P) was placed in a band 3-4 cm below the soil surface (Table 3). The soil inoculum from a pine plantation located at Berthierville, Quebec, was mixed in the 4 top cm of soil at time of planting the germinated seeds as described in experiment 3. On January 9, 1970, they were trans— planted in the pots when radicles were 0.5 to 1.0 cm long. The soil moisture level in each pot was periodic— ally adjusted with distilled water to approximately 20 per cent by weight as determined by weighing the pots. Several pairs of fluorescent lights were arranged parallel in the center of the plant bed 64.0 cm from the plant foliage level. These lights were automatically controlled and synchronized with the daily photoperiod to provide artificial light for a 16 hour period per day. This factorial arrangement of treatments was set in a randomized complete block design with 4 replications. After 19 weeks we collected the seedlings and made the same measurements as in experiment 3. Table 3. 35 Phosphorus and forest soil inoculum treatments used in Experiment 4.‘ No Treatment H 2 Soil 3 Soil 4 Soil 6 Soil 7 Soil 8 Soil 10 Soil 11 Soil 12 Soil 13 14 Soil 15 Soil 16 Soil 17 18 Soil 19 Soil 20 Soil Control inoculum inoculum inoculum inoculum inoculum inoculum inoculum inoculum inoculum inoculum inoculum inoculum inoculum inoculum inoculum (3.0 (6.0 (9.0 (3.0 (6.0 (9.0 (3.0 (6.0 (9.0 (3.0 ~(5.0 (9.0 (3.0 (6.0 (9.0 g/pot) g/pot) 9/pot) g/pot) g/pot) s/pot) 9/ pot) 9/pot) s/pot) 8/pot) s/pot). g/pot) s/pot) G/pot) s/pot) Superphosphate Superphosphate Superphosphate Superphosphate Superphosphate Superphosphate Superphosphate Superphosphate Superphosphate Superphosphate Superphosphate Superphosphate Superphosphate Superphosphate Superphosphate Superphosphate (224 (224 (224 (224 (448 (448 (448 (448 (672 (672 (672 (672 (896 (896 (896 (896 kg kg kg kg k9 kg kg kg kg kg kg kg kg k9 kg P/ha) P/ha) P/ha) P/ha) P/ ha ) P/ha) P/ha) P/ha) P/ha) P/ha) .R/ha) P/ha) P/ha) P/ha) P/ha) P/ha) 36 Field (Experiment 5) Since in experiment 4 we thought not to have attained the optimum growth of red pine and white spruce seedlings, we set up a field experiment with higher rates of P at the Ber- thierville nursery in Quebec in the Spring of 1970. The fer- tility of the sandy loam nursery soil is outlined below. ——‘—- L Available Nutrients (ppm) Organic pH matter _ _ % NH4 N N03 N P K Ca Mg 5.0 4.5 3.0 1.6 122 122 325 45 A complete randomized block design with four repe— titions was used. The P fertilization (Table 4) was applied on May 21. Phosphorus as triple superphosphate was incorporated in the first four inches of seedbed soil. Methyl bromide (0.9 kg per 9.3 square meters) was applied to the soil on the same day. Stratified seeds were sown on June 4. Nitrogen fertilization, applied at a rate of 56 kg N/ha to all plots, was delayed to July 17. Total precipitation at the field plot site for the 1970 growing season was normal as compared to the eleven precedent years. April and May precipitation were normal, 37 Table 4. ’Rates of phosphorus applied to nursery soil (Experiment 5). Treatment P no kg/ha 1 _ - _ 2 336 3 672 4 1008 5 1344 6 1680 but June, July and August was below normal and we supplied by overhead irrigation. Meanwhile in September the precip- itation was more than twice the amount of the precedent years. A comparison between the 1970 season and the period of record is shown below. Growing Season Precipitation (cm) April May June July August Sept. Total Period of Record 6.4 6.4 7.5 9.1 9.3 7.2 45.9 (1959 - 1969) 1970 Growing 5.9 6.9 4.3 5.4 6.3 16.5 45.3 season 38 During the season observations were made on seedling growth. On October 7, 10 seedlings were collected in every plot and their length was measured. We also recorded their dry shoot and dry root weights. Moreover the seedling shoots were analysed for P concentration using a method modified from Jackson (1958). Our data were submitted to analysis.of variance and regression. CHAPTER IV RESULTS AND DISCUSSION The influence of eradicants on mycorrhiza forming fungi varies considerably depending on the nature and con— centration of chemicals. A better growth of conifer seed- lings is often related to soil fumigation (Wright, 1964), but it also happens that fumigants are shown to reduce drastically the growth of conifer seedlings and preclude the uptake of even available nutrient elements (Henderson and Stone, 1970). This research presents experimental evidence that fumigation significantly affects the myco- trophic mechanism of nursery soils and subsequently P up- take by conifer seedlings. The influence of N and P fer- tilization, inoculation with pure cultures of mycorrhizal fungi or forest soil were evaluated for their effect on the growth of conifer seedlings raised in fumigated soil. The experiments were conducted in the greenhouse, field and controlled environment chambers. 39 40 A— NITROGEN AND PHOSPHORUS FERTILIZATION OF FUMIGATED OR HEAT STERILIZED NURSERY SOIL - GREENHOUSE (1) AND FIELD (2) EXPERIMENTS l- Mogphological CharacteristiCs Greenhouse - About 5 weeks after germination P deficiency symptoms appeared on white spruce and red pine seedlings growing in fumigated pots not having received any .supplemental P. This reddish purple discoloration lasted and intensified until the end of the growth period. The discoloration of the seedlings was accompanied by a severe decrease in growth (Tables 5, 6). In pots having received rock phosphate or bone meal this reddish purple discolora- tion appeared at the beginning, but disappeared with the lengthening of the period. Healthy and dark green seedlings grew in pots treated with superphosphate fertilizer. The growth and quality of seedlings, grown in ster- ilized soil without supplemental P, were not improved by the addition of N (Figure 1). In pots without supplemental N the addition of P significantly increased the size and quality of seedlings independently of the P source. Meanwhile in un- sterilized soil the addition of either N or P had not sig- nificant influence on morphological characteristics of red pine and white spruce seedlings (Tables 5, 6). 41 8.88 8.88 8.8 8.88 8.84 8.8 8.88 8.44 8.8 68848 8.88 8.88 8.8 8.88 8.84 8.8 8.88 8.88 8.8 A88.8 3 8.88888 8.88 8.888 4.4 8.88 8.888 4.4 8.88 8.888 8.4 8888 488 8.888 8.488 8.4 8.88 8.888 8.4 8.88 8.848 8.4 888:8 482 8.888 8.888 8.4 8.48 8.888 8.4 8.48 8.888 8.4 8888 482 8.84 8.88 8.8 8.84 8.88 8.8 8.88 8.888 4.4 8 482 8.88 8.488 8.4 8.88 8.888 8.4 8.84 8.88 8.4 8888 882 8.888 8.888 8.4 8.88 8.848 4.4 8.88 8.888 4.4 888:8 882 8.888 8.888 8.4 8.88 8.848 8.4 8.88 8.488 8.4 8888 882 8.88 8.88 8.8 8.84 8.88 8.8 8.84 8.888 8.4 8 882 8.88 8.888 8.4 8.88 8.888 8.4 8.88 8.88 8.4 8:88 8 8.88 8.888 8.4 8.48 8.88 8.4 8.88 8.88 8.8 88888 8 8.88 8.888 8.4 8.88 8.888 8.8 8.84 8.88 8.8 8888 8 8.84 8.88 8.8 8.88 8.88, 8.8 8.88 8.88 8.8 8 8 me me EU me me EU me me EU A Z 8888 88888 888888 8888 88888 888888 8888 88888 888888 888H83_888 emons 888 888883 888 88H288m 988882 2888888888888 8888 mmmmmmm. 888Hawe888 AH unmeflummxmw mmcflacmmm OCHQ OOH OHO 8x803 0H mo moaumflumuomumzo ”monOHoxmuOE no mucmfiummuu umnaawuuwm Dcm moaemwflafluwwm H808 mo uomwmm .m OHQMB 42 4.88 8.88 4.8 8.88 4.84 8.8 8.88 8.44 8.8 888.8 8.88 4.88 8.8 8.88 8.48 8.8 4.8 8.88 8.8 888.8 3 8.88888 8.48 8.888 4.4 8.84 8.888 8.4 8.88 8.88 8.8 8888 488 8.48 8.848 8.4 8.88 8.888 8.4 8.88 8.88 8.4 88888 488 4 8.88 8.888 8.4 8.88 8.888 8.4 8.88 8.48 8.4 8888 82 8.88 8.88 8.8 8.88. 8.88 8.8 8.88. 8.88 .4.8 8 482“ 8.88 8.88 8.4 8.88 8.88 8.8 8.88 8.88 4 8 8888 882 8 8.88 8.888 4.4 8.84 8.888 8.4 8.88 8.88 8:4 88888 82 8.44 8.88 4.4 8.84 8.88 4.4 8.88 8.88 8.8 8888 882 8.88 8.88 8.8 8.8 8.88 8.8 8.88 8.48 8.8 8 882 8.84 8.88 8.8 8.88 8.88 4.8 8.88 8.88 4.8 8888 8 8.88 8.88 4.8 8.84 8.88 .4.8 8.88 8.88 8.8 88888 8 8.84 8.88 8.8 8.84 8.48 8.8 8.88 8.88 8.8 8888 8 8.88 8.88 8.8 8.8 8.88 8.8 8.88 8.88 8.8 8 8 .lmE m8 EU me me so me me so m z 8888 88888 888888 8888 88888 888888 8888 88888 888888 888883 888 888883 888 888883 888 88888mm.888888 2888888888888 8888 8888288 8888888888 A8 pcmeflummxmv mmcflacmmm monumm mafia? @HO wxwm3 88 mo moaumfluwuomumnu HEUHmOHofimuOE co wucwauwmuu uwuflafluumw cam cowumN8HHHmwm 8808 no powwmm .8 OHQmB 43 , 4 8 4 . 8.888888888888888 8.4883485 n m .48 88888888 n 8 .888 8 88 8 828 n 88 .88 umpcmEu8088V uQmEQOHw>¢© mQHHUmmm Apsmfluv monumm mpHSB Ucm Auwwaw mafia Umu UHO 88883 08 :0 80888668 A 6mm 2 .coapmmflfism 8808 mUHEOHD.H%£#mE Mo powwmm .8 musmflm 44 Although significant main effects due to soil steri- lization occurred, the important features are the inter- actions between soil sterilization, N and P (Appendix Table 22). It is noteworthy to know that seedlings growing in sterilized soil with added P are better developed and have a greater shoot and root dry weight than those raised in un- treated soil. To promote growth of red pine and white spruce seedlings superphosphate seems to be a better source of P than either rock phosphate or bone meal. Superphosphate alone or combined with N treatments from either N03 or NH sources generally resulted in a better height growth and a greater shoot and root dry weight than with other treatments. Meanwhile NH4 sulfate and superphosphate appear to be the best combination for raising red pine and white spruce seed— lings. The shoots and roots of these seedlings were at least two times heavier than the control (Figure 2). Eigld - By the end of July P deficiency symp- toms appeared on white spruce seedlings growing in fumigated nursery sub-plots not having received any supplemental P. This reddish purple discoloration lasted and intensified un- til the end of the season. This phenomenon also appeared in sub-plots treated with rock phosphate or bone meal, but to a much lesser degree. Generally, the discoloration of WWW RED PINE "NH4 N N03 7///////////////////////////////////////////// O 2 l g E 2 § i “ 8‘- 2 .5 m [:I g 2 g 3 é é 2 :3: <3 2‘ .6: 9N‘lH9I3MAmiOOHS Figure 2. Influence of N sources and superphosphate addition on the shoot dry weight of 16 weeks old white spruce and red pine seedlings grown in methyl bromide fumigated soil (Experiment 1). 46 the seedlings was accompanied by decrease in growth. Healthy and dark green seedlings were growing in sub-plots treated with superphosphate fertilizer. The same observations apply to the red pine seedlings but the discoloration appeared later and was much less intense. First sampling date (14 weeksl - Although, as in the greenhouse experiment, significant main effects occurred, the important features are the interaction between soil fumigation and P, and N and P (Appendix, Table 23). Seedlings growing in untreated soil were generally not significantly affected by the addition of N or P. The addition of rock phosphate or superphosphate to fumigated soil was related to a significant increase in seedling growth and development, while bone meal did not influence them. Still larger seedlings were obtained when N and P were added together to the same soil. As reported for the greenhouse experiment, super- phosphate alone or combined with N treatments from either N03 or NH4 sources resulted in a better height growth and a greater dry weight of shoots and roots than with any other treatments (Tables 7, 8). Again NH4 sulfate and superphos- phate appeared to be the best combination for raising red pine and white spruce seedlings (Figure 3). The shoots of 47 H.m ©.o m.m m.vH m.o ¢.o u.mfl v.0 ”Ho.w m.v m o m.v m.mu m.o v.m m.oH m.o “mo.v 3 m.>mxnm s.mfl m m p.ma o.mv H.v m.oH m.sm n.m «com m.mm o.mm o.mm s.v ~.sa m.~m H.¢ ummsm o.mm m.qm m.ms m.v s.su p.mv o.¢ xoom o.mH m.mH m.mm m.¢ o.ma o.~m m.m o N.mH m.ma m.mv m.m o.oH m.mm o.m mcom m.ma o.Hm s.on «.4 m.mH o.mv >.m nomsm N.ma m.oa s.om o.v o.mH s.nm m.m xoom «.ma r.ofi m.am m.v o.mH o.Hv n.m o m.vg o.ma 0.4m H.v o.OH 0.0m m.m «mom N.HN n.om m.mw m.v m.mu r.vv m.m ummsm N.sH m.ma n.mv H.v o.sa «.mv m.m xuom m.ma m.nn N.mv o.v N.ma o.mv m.m 0 me m& we Eu me me 80 Room. woom moomm mmon meow 900mm smemm m summmszmn mmonz man wmmHmz man mnHzomm ammsmz nom~2do mmNHuHmmmm .AN pmmefluwmxmu HMUflmOHOLQMOE no mucmEpmmuu umuaafluumw one floaummHEDM MHom mo uummwm . mmcwaomwm mafia own oao mxmmB «a mo muwumfluwuomumflo 48 ~.v o.HH m.o m.m m.HH m.o ~.m m.s o.o Ago.o m.m m.m 5.0 M.m o.m p.o m.H H.m m.o Amo.v 3 m.»wxse ¢ m.m m.oa H.m o.m m.vm m.m «.5 ~.m~ v.m mcom ma v m.ma n.vm m.v m.HH o.mm H.m m.m r.mm o.v nmmsm mz v m.m m.om m.m n.m n.mm s.m ~.m o.o~ H.m room mz ¢ o. m m. 3 m. m m. m m. 3 m. m m. m m. 3 m. m 0 ma. 0.4 o.vH m.m m.o m.ma o.m m.m m.mH H.m waom moz m o.m m.nm m.v o.oH n.Hv m.¢ m.m m.om ~.m ummsm oz m m.m m.ma o.m m.o o.mH o.m s.¢ o.mH m.m xoom oz o.v m.mH o.m s.v o.mH H.m m.m m.mH m.m o moz 0.0 o.o~ m.m o.n s.mm ¢.m s.m m.mm m.m muom o o.m «.mm m.¢ m.HH m.Hv m.v m.m o.mm o.m ummsm o m.o o.mH N.m m.s o.mH ~.m m.m o.mH m.m xoom o ~.m m.vH m.m m.m m.ma m.~ ~.m s.MH m.m o c me me EU me me So me we EU Boom Hoomm emonm moom 900mm emoumm Boom soomm mme,. m z smonz_wmn smonz sma Hmonz wan sawmp monomm qwmn, somHZOU mmNHuHemmm .mm unmsaummxmu mmnflfiommm monumm oufi£3 6H0 mxmw3 vH mo moflumfluwuomnmfio HMUHmoHOSQHOE so muumebmwuu umuflflflpumm pom godpmmMEDw HfiOm mo vowmmm .m manna 49 A.mpm£mmogmuomsm + VOmNAvmzv n HH twpmxmmowmnmmsm + mozm2vm mm wwmnmmonmummsm u m .Houumoo mzv u m .mvmammonmuwmsm u IHwHflunwm I owflomwm cmummHESH Aumev wQHQ own no npsoum n H .uSmHu .w+w£mmonmuomsm + om“ may n HH m .Houumoo n H +uwH .munmeummqu hummusn oHHH> ooHEouQ Hhsumfi QH csoum Aunmwuv monumm oufia3 can uoou cam uooaw mg“ no :oHuHoom m cum 2 mo vacuum .m wuzmfim 50 these seedlings were two to four times heavier and the roots three times heavier than the control (Figure 4). Second sampling date (17 months) - White spruce seedlings grown in fumigated soil showed a much better growth and a healthier appearance than those grown in untreated soil. The main effects of experimental factors are highly signif- icant (Appendix Table 24) and only the interaction of fumiga- tion and phosphorus application is showing high significance (0.01 level). At the end of the second growing season seedlings were dark green, sturdy (Figure 5) and showed a balanced root system. We can see the beneficial effect of N and mainly P fertilization on the seedling development. Looking at N sources the superiority of NH4 over NO3 when used in combina- tion with P becomes much more evident than at the l-O stage (Table 9). Indeed the addition of N alone did not improve seedling growth. Rock phosphate did improve the growth, but not significantly; meanwhile superphosphate increased the shoot or root seedling dry weight to ten times when the soil had been fumigated with vapam. In unfumigated soil the growth increase of superphosphate addition was not so large, about three times the control (Figure 6). V §§ NOFHOS*KRUS His 13£ERPHO§*MWE //////////////////// RED PWEI \NHHEISPRUCE / ////////m 51 ///////// ////////////////////////////////////////// ’//////////////////////////////////4 W W ///////////////A ’////////////A W '////////////. W/ loo—- 80— l l 20- 8 9 9w-1H9I3M mo locus V I /E K) 29 O 2 /é’ Figure 4. Influence of N sources and superphosphate addition on the shoot dry weight of 14 weeks old white spruce and red pine seedlings grown in methyl bromide fumigated seedbed (Experiment 2). . Sou wuwmuzc wHH mmCHHomwm monumm w v m.v unoo u m “mamfimm0£muwmsm + 0m r mzw n d “mummfipmmnwv H>H0H£uuwm I U vans onm m0 a» wnpwmm owpmmflssm mofifioun Hhflpwfi cw c3oum 30Hm poonm so GOHuHomm m can Z we vummmm .m wusmHm 53 Hom mHHH s.o mum Hms m.m AHo.V mmm mmm m.m mmm Hmo «.4 Amo.v3 m.smxps mHoH mmom ¢.mm mvw mmoH m.HH ummsm 4m2 omm mmm s.HH mom mmo m.m xoom wmz mmH mmm v.m mmm omm ¢.m o vmz mom onH m.mH ova mmm H.m umosm moz mom omo H.oH va mmv H.» xoom moz 4m omm o.~ omm mmm m.@ o moz mam HHNH «.mH mam Nov m.» uwmsm o omH wom m.OH ooH Ham m.o room o 40H nHm m.m mmH Hem o.h o 0 m8 . m9 EU .me we EU poem woosm “smHmm boom noosm usmHom m z emoHummem mmonz,»mo 24m<> uommzoo mmNHuHemmm .Am pcmEHummxmv msommmm ngOHm ozu umumm meHHmem monhmm mUH£3 mo moHumHumuomummu HmonoHoflmuos co muouomw HousmEHnmmxm mo wononmsH .m mHQMB 54 \ s NNo3 PSUPER V ‘\\ NNH4 PROCK \ \\“ ”NH4 pSUPER D No Po s N, .m WWW/Wfl/ /////// ///////////////// W/I/llllllll /////////////// 3000- ZKXL— 2000— 5004 SW "J.HSI3M mo lOOHS |OOO— 500— VAPAM CONTROL Influence of soil fumigation, N and P addition on the shoot dry weight of 2-0 white spruce seedlings (Experiment 2). Figure 6. 55 Discussion - Thorough soil sterilization with methyl bromide, vapam, or heat, duplicated in greenhouse and field experiments the adverse effects on seedling growth ob- served in routine nursery practice. In both experiments we reached statistical significance. The severe decrease in growth and seedling develop— ment following soil sterilization could in part be attributed to the fact that this soil treatment had killed most soil microorganisms and fungi capable of forming symbiont associa- tion with seedling short roots. Mycorrhizal formation having been restricted, there was a lesser root surface for absorp- tion, and then P absorption and growth could have been de- creased (Hatch, 1937). Henderson and Stone (1970) observed that both growth and P content of non-mycorrhizal coniferOUS seedlings grown in fumigated soil without P fertilization were less than any other treatments: they attributed this phenomenon to the absence of mycorrhizae. It seems that, in the absence of mycorrhizal develOpment of roots, the young seedlings can only absorb P when present in inorganic form at high concentration in the soil solution. From the three P sources used, only the superphos- phate addition was consistently related to a very good growth rate and a dark green seedling color. Meanwhile at sampling time, despite a fairly good root development, we observed few 56 if any mycorrhizae. It is possible that mycorrhizae were still not visible at that time. Larger seedlings could also result from a higher soil fertility level which can provide adequate nutrition even in the absence of mycorrhizae (Howe and Clifford, 1962; Wright, 1964; Henderson and Stone, 1970). Nitrogen alone had very little influence on seed- lings grown in sterilized soil. But addition of N and P were related to larger seedlings than P alone. Furthermore NH4-N was consistently a better source of N than NO3-N. At pH around 5.0 the NH4 form of N is prdbably more available than the N03 form to conifer seedlings (McFee and Stone, 1968). The seedlings may absorb NH4-N faster than NO -N, or 3 because N is lost by leaching in the N03 form. It becomes apparent that the best combination would be a fumigated soil, a NH4 and a superphosphate addition. Seedlings produced in this manner compare favorably in size with standards set by Armson and Carman (1961). Comparison with the unfumigated controls shows that following fumigation harmful pathogens are eliminated and seedlings growing in soil with adequate amounts of N and P make good first season growth and well formed buds for the next season. Since in conifers the growth conditions prevailing in one season determine to a large part the next 57 year's growth, the preforming of a good shoot and terminal bud is prerequisite to a healthy seedling. 2- Mineral nutrient concentration of shoot Greenhouse - The significance of mineral nutrient concentration of seedling shoot tissue seemed to be due not only to the main effects of the experimental factors but also to their interaction (Appendix Table 25). In untreated soil red pine and white spruce shoot tissue had a significant lower N percentage than those raised in sterilized seedbeds (Tables 10,11). The seedlings grown in sterilized soil not receiving any N also had a low level of this element in their shoot tissue. The addition of N as NH4 or NO3 sources significantly increased the shoot tissue concentration of this element. In the white spruce and red pine seedlings there is a significant interaction of P and N fertilizers. Indeed the addition of either super- phosphate or rock phosphate is related to a sensible decrease of the N level in shoot tissue (Figure 7). This could be related to a better development of the seedlings accompanied by a dilution of N concentration in shoot tissue. The addition of any P source is related to a signif- icant increase of P concentration in shoot tissue, either for wmo. mma. hm. mma. who. #0. mma. mm. mad. mma. 0m.H NAM. mm.H mmm. Eva. hm.H wma. mm.a NNN., mma. ©®.H fioa. mm.H Hmm. omo. HN.N omo. mV.N mma. hmo. wN.N MHH. ©N.N wow. mma. mm.H 00H. vm.H mod. Boa. hv.a FMH. ©h.H VHN. Nmo. hN.N omo. Nm.N mwa. mmo. m®.H mOH. mm.H fiON.. NNH. ©H.H vmm. NO.H mwm. mma. ©O.H vMM. NO.H mNN. moo. m®.M .DO. mmoa HON. X. R INN x x A Z 2 m MOHEOmm mehmz -104 woznmmHonmmm sewn mo. xHo.o mm. Amo.v 3 m.>mon . v.. mm H mcom m7 4 - mv.H ummsm m7 . v - mm 0 300% m? mm.H o emz m - mm.H mcom oz n¢.H ummsm moz . m 4 mo H xoom or m mV.H 0 02 mm.“ mqom o mo.H ummsm o km.o room o em.o o o n z m z Homwzoo mmNHHHHmmm II"; 1‘ 0.... .HH unwEHmexmv mmCHHpmmm wCHm own UHO mxww3 0H wo Hfiw COHumuucmocou msucxmmCSQ pom GomouuHa “OOHm no mufiwEummuu HwNHHHuHmm pan :oHuMNHHHHmum HHOm mo uommmm .oH mHfinH 59 woo. Ho. mmH. om. oso. m¢.o xHo.v vmo. Hm. mmH. Ne. mmo. om.o “mo.vz m.>mxpu va. om.H mmH. ms.H omH. mo.H maom vmz mmH. mm.H moH. om.H mam. mm.H ummsm .vmz mmH. mm.H mmH. mm.H eHm. me.H room vmz moo. mm.m MOH. mv.~ mmHn om.H o vmz moH. mm.H smH. em.H poH. mo.H «com moz Hem. me.H How. we.H Hmm. mm.H uwmsm moz omm. me.H mmH. mm.H smH. nm.H xuom moz Non. HN.N moo. mH.N mNH. om.H o moz mmH. me.H NHH. mm.H mmH. o~.H ocom o HHN. mo.H meH. em.o mom. hm.o uwmsm o omH. oo.H oon. mm.o Hmmw oo.H xuom o omo. mo.m moH. mo.m mom. sm.o o o m z m z a z, m z monomm qwmemz momnemHonmnm amen onmnzoo smmHuHHmmm .HH psmEHuomxmv mmmHHoowm muommm «MHSB oHo mxwmz 9H mo “XV GOHumuunmosoo mononmwogm osm mwmoupHo uoogm no mpnmeummuu umNHHHunww tom sOHumuHHHHmum HHOm mo powmmm .HH wHDmH 60 ’////[///////////////////1 ’/////////////////////// '//////////////////l . ’///////////////////// «1 200'“ (%0 lOOHShflIWMHRMNBQWXJhEDOEMN SUPER Influence of P and N fertilization on the N concentration Figure 7. (%) in shoot tissue of 16 weeks old white spruce seedlings (Experiment 1). 61 red pine or white spruce seedlings. The greatest increase was obtained with rock phosphate and superphosphate, as shown below. P Sources Phosphorus Concentration in Shoot (% Drnyeight) Red Pine White Spruce None .116* .123* Rock phosphate .174 .192 Superphosphate .174 .211 Bone meal .145 .158 * Means of 36 determinations. Following soil sterilization, the P concentration of seedling shoot tissue was lower than for seedlings grown in untreated soil. This reduction of P uptake was really severe when no P fertilizer was added to the soil (Figure 8). The addition of rock phosphate or superphosphate to sterilized soil brought a net increase of the shoot P concentration and a good rate of growth. This appears to be a typical effect of soil fumigation or sterilization on the uptake of P by young conifer seedlings. Red pine and white spruce seed- lings grown in untreated soil showed a P level considered sufficient for an optimum growth, even when no P was added to the soil. 0.0 I x 8 a"! W 7/////////////////////////////////////////////////////////////// ’/////////////////////////// 62 //////////////////////. WWW/WWW //////////////////// ///////////////////////////////// \.\\ a § cf’ $9 7//////////I///////// //////////////////////////////////////////////////////////////// 7////////////////////////// /////////////////////////////2 W ' § V//////////////////////////////////////////////////////// 025- T l 9 To (%) lOOHS NI NOLLvumaoNoo SflHOHdSOl-H OJOn 0054 STERILIZATIO‘J HEAT Influence of soil sterilization and P sources on P BROMIDE METHYL CONTROL Figure 8. concentration (%) in shoot of 16 weeks old white spruce seedlings (Experiment 1). 63 Field — The significance of mineral nutrient concentration of seedling (14 weeks old) shoot tissue seemed to relate more to the main effects of the experimental fac— tors than to their interaction (Appendix Table 26). Soil sterilization was related to greater accumulation of N in shoot tissue, whereas it significantly decreased the P con- centration. The addition of N as NH4 significantly increased the shoot tissue concentration of this element. Indeed we had a greater N concentration following a NH4 than a NO3-N addition (Figure 9). The addition of any P source to fumigated soil is related to a significant increase of P shoot tissue concen- tration either for red pine or white spruce seedlings. The greatest increase was obtained with superphosphate. Rock phosphate or bone meal addition brought a much lower P con- centration (Figure 10). This fact supports the visual ob- servation of a P deficiency in seedlings growing in plots with no supplemental P or treated with rock phosphate or bone meal. The non-deficiency level was reached only when super- phosphate was added to the fumigated soil (Tables 12, 13). The addition of superphosphate to soil is related to a significant increase of foliar K concentration for both red pine and white spruce seedlings. WHITE SPRUCE RED PINE 64 WWW ’////////////////// 7//////////////////////////////////////////////////A ’/,////////////////////x W é ' ('3 cl) #3: § “2 9 I50- (%) lOOHS NI NOIlVHlNHDNOD NBDOHIIN NITROGEN SOURCES Influence of N sources on N concentration (%) in shoot of 14 weeks old red pine and white spruce seedlings (Experiment 2). Figure 9. WHITE SPRUCE RED PINE 65 WW 7///////// //////////// 7//////////////M /////////////////// 7//////// / l 0300- (1250-# 0200~ (LISO-fi (LIOO—i 6%)MXWSAHADUVHUEDWDD‘9“flMdfl}H O.C)50--I Influence of P sources on P concentration (%) in shoot of Figure 10. 14 weeks old red pine and white spruce seedlings (Experiment 2)- 66 mm. mm. Hvo. ow. mmo. mm. mm. moo. mo. AHo.V om. emo. mm. as. mmo. mm. om. who. om. Amo.oz m.>oxse oom. msH. oo.~ moo. moH. mo.m who. mHm. mo.~ mcom vmz oak. mew. vo.m ooh. mom. om.~ moo. Hmm. mv.~ “mono «oz nmo. mmH. mo.m moo. NoH. oo.~ oso. moH. oo.~ xuom . vmz cmo. ka. me.m moo. mmH. mo.m moo. moH. eo.m o «m2 moo. ooH. mm.m oso. HoH. Hs.m ooh. pom. o~.m mcom moz ooh. mom. me.~ «on. mHm. om.~ ome. vow. sH.~ “mono moz ooo. mMH. Hm.~ ooo. oeH. e¢.~ sow. moH. k~.m xoom moz pom. omH. Hm.~ who. emH. Hm.~ mom. msH. vo.~ o moz omo. mmH. Ho.m moo. moH. oo.m ooo. omm. HH.~ wcom o ems. on. om.m ooo. HHN. mm.m poo. oom. mo.~ modem 0 moo. mmH. mm.m mHo. meH. om.m smo. on. mm.H xoom 0 who. eMH. mm.m moo. omH. mm.~ moo. sHm. Hm.H o o X R X X. x. X. X _X R x Ilmll. 2 M m z :z m z m z z monomm Hzmsmz Hommzoo mmNHHHemmm .Hm ucmEHummxmv mmcHHomom msHm own 6H0 wxomB wH mo ARV COHumnucoogoo Ezflwmmpom one msuosm Imogm .cmmouywc uoonm no mucmeummuw umNHHHuumm pom coHummHEom HHOm mo uommmm .NH oHQMH 67 mm. oho. om. om. ooH. om. om. oso. mm. xHo.o oH. ooo. mm. om. moo. om. mo. woo. on. ”mo.o3 m.smxss so.o on. oo.~ oo.o ohm. oH.m oo.H Hom. sm.~ «com «on oH.H «mm. ss.m HH.H on. Hs.m vo.o oom. mm.~ ummsm omz oo.H mam. mn.m oo.o omH. on.m mo.o mom. s¢.~ zoom emz om.o ooH. mo.m kn.o moH. s¢.m oo.o How. on.m o vmz oo.o onH. es.~ on.o Hmm. Hm.~ mo.o mom. oH.~ mqom moz om.H mom. No.m oo.H mom. sm.~ Ho.o on. oo.m mango moz om.o osH. H~.N mo.o mmH. o~.m mo.o Hmm. oo.~ scam moz mo.o omH. vH.m mm.o ooH. om.~ mo.o mom. oH.m o moz mo.o oom. so.~ mm.o omm. on.~ MH.H on. oo.~ mcom o om.H oom. mm.m oo.H mom. oo.m mo.o omm. oo.H umdsm o mo.H moH. om.m mm.o ona. mH.N Ho.H How. so.~ zoom o mo.o ovH. Ho.“ mm.o ooH. so.m mo.H mum. oo.~ o o X X .x. x. X x. .x. R. .x. M IIMII. Z M A Z M m 2 m 2 z monomm Hyman: oomazoo mmuHoHammm Imonm .cwmouuHc uoonm no mucoEpmmup HmNHHHuumw cam coHummHEsm HHom mo powwmm .AN unmefluwmxmv mmcHHommm oosumm muH£3 oHo mxmoz vH mo AXV soHuwuucwocoo EsHmmmuom pom .mSHOHQ .MH mHQMB 68 Discussion - In both experiments a greater amount of N was accumulated by seedlings growing in sterilized soil than in untreated soil. This could be in part due to the fact that after fumigation N accumulates in the soil as NH4 and this form of N is easily taken up by conifer seedlings. This NH4 accumulation could be related to a near annihilation of the soil nitrifier population by sterilization. Both tree species appeared to prefer NH4-N over NOB-N either for growth or N uptake in their shoot tissue. The increase in shoot N content following soil sterilization seems to be also related to a better growth of seedlings following P fertilization in sterilized soil than in untreated soil. The red pine and white spruce seedlings showing evi— dent P deficiency symptoms gave a P concentration as low as .090 per cent when grown in the greenhouse, although those raised in the nursery beds showed a little higher P content for the same symptoms. Swan (1960) reported evident P deficiency symptoms ' at 0.070 per cent in shoots of Pinus banksiana and at 0.100 per cent in Picea_glauca and P. mariana raised in greenhouse. Fowells and Krauss (1959) reported symptoms in Pinus taeda and P. virginiana at 0.100 per cent P in the leaves. Ingestad (1962) found that deficiency symptoms are generally related to a foliar P concentration of 0.060 - 0.090 for 69 pines and 0.050 - 0.110 for spruces. On the other hand, Armson and Carman (1961) reported as deficient a P level lower than .200 per cent for red pine and .250 per cent for white spruce seedlings raised in nursery beds. Our data agree very well with findings of these workers. The very low level of P in shoot tissue of seedlings growing in sterilized soil, without addition of P, was associated with a very poor seedling development and an ab- sence of lateral and short roots. They showed a reddish purple discoloration of their foliage which is symptomatic of P deficiency. It is probable that soil sterilization severely reduced mycorrhizal fungi and other soil micro- organisms necessary to a good availability and uptake of mineral nutrients (Iyer, Chesters and Wilde, 1968). Mechanisms which make nutrients available to plant roots are: (1) Root interception, (2) mass-flow, and (3) diffusion. The concentration of soluble P in the soil solution is always low except close to sites of applied fer- tilizer. Moreover P is not normally considered to be a mobile nutrient and mass-flow is known to supply less than one per cent of the P needed by corn (Barber, 1964). Ap- parently, the rate of diffusion of P varies with soil mois- ture and is greater when more water is present. This makes 70 more P available in the root zone where root interception and contact feeding take place. . What has been learnt about pine root distribution and what is known about the way P moves in the soil give reason to believe that mycorrhizae do in fact help with P nutrition. The absorbing portion of a growing root remains functional for only 5-10 days, but mycorrhizae continue to absorb nutrients for a much longer period - possibly for as long as a year. under these circumstances P from a solid particle will diffuse much further whatever the soil condi- tions are. The exploitation of soil reserves is thereby increased substantially. Moreover the hyphae, being much thinner than root hairs, can insinuate themselves into finer soil pores and this does increase the degree of soil ex- ploitation. It is known that mycorrhizal fungi can store large amounts of P. This facility may be important in trapping P when large amounts suddenly become available, such as after a P fertilizer has been applied, and making it available to the plant at a later stage. Ekperiments have also shown that mycorrhizae can absorb a number of organic compounds not normally taken up by higher plants. The fumigation must have also had an effect on other rhizosphere microorganisms. The interactions of soil 71 fumigation, rhizosphere microorganisms on the availability of soil P for plant uptake could here be related to a lesser rate of seedling growth and a lower P content of shoot tis— sue. Indeed Gerretsen (1948), studying the influence of rhizosphere microorganisms on phosphate uptake in plants grew a variety of crop plants in sterilized and non- sterilized soil, to which various insoluble phosphates were added. He observed a greater phosphate absorption in the plants growing in non—sterilized soil. Phosphorus as adenosine triphosphate and numerous phosphorylated products participates in nearly all syn— thetic reactions of the plant. It is essential for the development of meristematic tissues and it is associated with the general process of respiration. We can infer that any reduction in P absorption by coniferous seedlings due to unavailability of the P source and root surface factors will have a strong influence on seedling growth and the con- centration of shoot P. The level of K in shoot tissue is in the range set by Armson and Carman (1961) for an optimum growth of 1-0 conifer seedlings. The greater K uptake following superphosphate soil addition is probably related to the Ca content of this fertilizer. Superphosphate addition significantly increased the Ca available in soil. Jacobson et a1 (1961) reported an 72 increased K absorption by barley (Hordeum vulgare), corn (Zea mayg), and sunflower (Helianthus annuus) roots, when Ca was present in the nutrient solution. 3- Soil Fumigation, Phosphorus Addition and Seed- ling Survival - As reported by other workers (Howe and Clifford, 1962; Wright, 1964) soil fumigation greatly in- creased first year survival (Table 14). This increase was particularly high when the test species was red pine. In unfumigated soil high losses from damping-off were usually observed. The addition of P did not affect survival, except that addition of bone meal was significantly correlated with a reduced survival of seedlings. Bone meal seemed to have been related to a lower germination and a high damping-off mortality either for red pine and white spruce. Wahlenberg (1930) also reported that addition of bone meal or dried blood caused increased damping-off of conifer seedlings. The addition of superphosphate slightly decreased white spruce stocking. Benzian (1965), summing up experiments covering many soils and seasons in English nurseries reported that superphosphate had been found safe although occasionally survival can be slightly decreased by phosphate applica- tions. 73 Table 14. Effect of experimental factors on first year survival of red pine and white spruce seedlings (Experiment 2). RED PINE WHITE SPRUCE Soil fumigants No seedlings/ No seedlings/ linear meter linear meter None 8 52 Methyl bromide 24 79 Vapam 3O 67 Tukey's w(.05) 4 16 (.01) 6 23 P. sources 0 25 86 Rock 26 81 Super 23 77 Bone 8 26 Tukey‘s w(.05) 3 6 (.01) 4 8 74 4- Effects of Soil Fumigation on Soil Character— istics - Soil reaction has an important effect on growth of most plants since it alters availability of nutrients and the constitution of soil micro-flora and fauna. Soil fumi— gation significantly increased the pH level in soil whereas here the addition of either N03 or NH4-H lowered this level (Appendix Table 27). The acidifying effect of NO3 cannot be easily explained, except by an unknown interaction. As foreseen NH4 sulfate addition significantly decreased soil pH (Table 15). The soil pH was not altered by superphos- phate or rock phosphate sources. The addition of bone meal markedly reduced soil acidity. This effect is probably due to the high organic and Ca content of this P source. As reported by others (Good and Carter, 1965; Driessche, 1969), our nursery soil fumigation either with methyl bromide or vapam delayed nitrification for the first growing season and was characterized by NH4 accumulation (Table 15). It is generally recognized that the nitrifiers are among the most sensitive of soil bacterial flora to in- jury by fumigant chemicals (Martin and Pratt, 1958). Wolcott et al (1960) observed that the nitrifying activity of a muck soil was sharply reduced by fumigation with Telone at 385 liters per hectare. There was an 8-week lag before a signif- icant recovery occurred. Winfree and Cox (1958) reported 75 .mQOHumcHEuwuwo om mo mammz AU mcoHumnHEwaoU mv mo mummz Am 0am mm 5H VVH mm mo o~,. .oo.-. , oo-.o,.hHo-o‘..... NH no mm mo oH mm so. Amo.v3 m.>mxse mo ooHH mom moH om omH o.o mcom Ho on HmmH Ham woo on em H.o ummsm Am om mom mom mmH om mo H.m xoom .m om oom mom oHH oo oo o.m 0:02 AH mmouzow monogamonm AU mH «NH om om Hm om ‘ . so. oH 4o oH mm 4H om oo. Hmo.o3 m.>mxss on okoH mmm oom ms 4mm o.o vmz Am oo mooH Hmm mom obs s ~.m moz Am on mmoH mam mom mm mo ¢.m 0:02 AH mooH50m comouqu Am om ,NHm no .ms w.oo. on .. .HH. oH mam so oo om om oo. mmo.v3 m.smxss oo sooH oom «mm mm mvH N.o smmm> Am Ho HNoH mom mom mo moH o.o moHeoun Hanna: Hm om ooHH omm oom omH no o.m «:02 AH ooHpmmweom A4 m3\mx m£\wx m£\mx m£\mx m£\mx m£\mx ............ m2 MD M m moz vmz mm Houomm HmucmEHummxm .AN unmEHummxmv HHom hummuss nH ESHmmnmme onm .EsHuHmo .Eszmmuom mHQmmmomSUxm onm .mmz ofim vmz .mm no mnouumm Hmucweflummxm mo mocmzHMQH .mH mHQme .msnonmmonm oHQmHHm>m HHom 76 that either chloropicrin (412 liters per ha.) or methyl bromide (.1 kg per square meter) caused an initial accumula- tion of NH4-N at the expense of NO3-N. This NH4 accumulation in treated nursery soil could be a beneficial effect of soil fumigation since conifer seedlings seem to have a better growth when N is available in NH4 form (McFee and Stone, 1968). The addition of P had little effect on NH4 or N03 level in soil except for bone meal which significantly in- creased the NH4 soil content. This is evidence that pro- teins in bone meal were rapidly decomposed. Superphosphate, made by treating raw rock phosphate with suitable amounts of sulfuric acid (Buckman and Brady, 1968) seems to be an effective and economical carrier of phosphatic acid for conifer seedling production. The amount of P available at the end of the season in the soil fertilized with 505 kg of P as superphosphate was as high as 564 kg of P. If we consider the amount in the soil before any P addition it seems as though the major part of this added P is in an available form at the end of the season. P also could have been made available from organic sources mineralized during the season. The efficiency of P fertilizers with regard to the P supply to plants depends on the extent, and for how long, the fertilizer is able to increase the availability 77 of P in the vicinity of the plant roots. In this experiment superphosphate increases the soil test for P much more than either rock phosphate or bone meal (Table 15). This supe- riority of superphosphate as a P source is also shown in the size of seedlings grown following the addition of this fer- tilizer to the soil; Experimental factors were related to a few signif- icant increases or decreases of exchangeable amounts of other nutrient elements. However to explain them would require additional experiments with these specific elements. B- INOCULATION OF METHYL BROMIDE OR VAPAM FUMIGATED NURSERY SOIL - GROWTH CHAMBER (EXPERIMENT 3). In this experiment both fumigants, methyl bromide and vapam, showed a very similar effect on morphological charac- teristics of red pine and white spruce seedlings (Tables 16, 17). Meanwhile the inoculation of fumigated nursery soil with forest soil was associated with a much better growth and development of seedlings than control or other inocula- tion with pure cultures. There were highly significant (0.01 level) differences for all the studied seedling character- istics for both test species (Appendix Table 28). The green and dry weights of the seedling shoots and roots grown in 78 H.m smH omo mmm oomm m.m AHo.o o.m om Hoe moo oooH o.o Amo.o3 m.>mxse o.o om mo moH ooH m.m oHuummuumu.muonmmHme o.o Hm mo soH mom o.o .moumHocmmm,moHHHoo o. o no oo 2: 2m 4. m mauochflm aaooooamo o.oH ooH ooo moo mmmm s.» HHom nmwuom o.o Hm mm emH HoH o.o maoz moommm msHmz o.om oom omo sooH smHm o.H AHo.V o.om ooH How new omoH H.H Amo.o3 m.>mx=e o.o oHH soH ooo ome o.o moousH.moHHH:m o.k ooo mmv ooo vaH H.m moHommou.comomoanm o.o mMH koH Hmo mmm o.o mamomoaou-muHoms¢ o.om mom Hoo omoH smwm o.o HHom ammuom o.o moH mom who mom o.o mcoz msz.omm X I 1.: I.I.I.I ImEI.I I I I Hm: ...... EU... .cowumHooozH muoom uuono HmNHpuuouxz boom uoosm uoom poono uronm Hocom wnmwmz Nun unmfi®3 Gwmuw l”[ E .Am ucwEHHmmxmv mmcHHoomm monumm muHSS ooo msHm own 0H0 mxooB 0H mo onumHumuomumno HmUHmoHoamHOE so GOHHMHDUOGH Hmozm HMNHHHHOUME pom uoHummHEsw HHOm onEouQ thwoe Mo “comma .wH anmE 79 o.HH mo pom o«« «omH H.m HHo.o o.o mo oo« HH« oooH o.« “mo.o3 m.smxse o.o oH «o Hm m«« o.o mHuummuumu muonmone o.o o« «m oo ooH H.m moumHscmum moHHHso o.o 0H mm mm mm m.~ oeuomwcmum EDUUOUOGGU «.«H 4«H moo «oo oo«« o.o HHoo powwow o.o oH Hm oo oo o.« oaoz moommm meHms «.«H mm omH ooo oo« o.o xHo.o o.o «« moH mom ooH o.o Amo.v3 m.»mxse o.o mo o«H Hoe ooo o.o momusH msHHHsm o.o om o«H oom omm o.o msHowmou somomoNHgm o.o mm HMH wmw mom m.¢ msoumwnzu muwcmfi< s.n« ooo o««H omsH mksm o.o HHom ammuom o.o moH ««H «we m«m o.o mnoz .1msz mom x n u u I u u u n u u u u u no mnoom unorm HmNHnuuoosz poem noonm noom “Mono nnoHom uflmfloz mum uanwB nmmuo .Hm psmEHuomxmv mmcHHomom monumm wuHHB mam qum own UHo mxmoB oH mo moHuwHumuumumao HmowmoH nonmuoe so GOHumHsuocH Hmnsm HoNHflnuoo>E pom COHummHEsm HHOm Emmm> mo Hommmm .wH GHQMH 80 the forest soil inoculated pots were two to ten times greater than those grown in control pots (Figure 11). The former seedlings were dark green and were showing a healthy condi- tion and a balanced rate of growth at sampling time, while the latter were showing a reddish purple discoloration of needles, and signs of P deficiency. unfortunately, the pure cultures of fungi inocula- tion failed except for a very few pots. In most pots in this eerie the seedlings showed a reddish purple discolora- tion of their needles and poor growth. One of four pots inoculated with pure culture of Rhizopogon roseolus was particularly responsive to this inoculation.: These red pine seedlings were well developed and comparable to those grown in forest soil inoculated pots and their roots showed numer- ous mycorrhizae. Failure to get a consistent response to pure culture inoculation may be eXplained in several ways. First it is known that mycorrhizae fungi are not competitive in the soil unless they are in symbiosis with tree roots. A few fungi (Ex. Trichoderma viride) quickly reinvade fumi- gated soil and outgrow other fungi for a certain period of time (Martin and Pratt, 1958). Soil inoculation and sowing of germinated seeds were done the same day. It probably todk too much time for the young rootlets to make contact 81 Au .AHouucoo u m “EDHDUOGH Hfiom HHOm owummflssw ooHEOHQ thuwe GH czoum Auamfluw mev oaHm own no £u3oum wflu no QOHuHoom EsHonnH HHOm n H "mucwfiumouev mosHmm wuHHB can umwwom mo uuowmm .HH mHsmHm with to C CORE SEC tit DI r +’ t’) de We th. pot I ‘ , iatlc mYfel were (I 82 with the chunks of mycorrhizal fungi. Seed radicles (0.25 to 0.5 cm) were not developed enough at sowing time and consequently there was a delay before these rootlets could secrete exudates or growth factors which stimulate mycor- rhizal fungi growth to envelop the rootlets with a dense sheat of hyphae: the first sequence in mycorrhizal forma- tion. Melin (1954) concluded that pine roots produce one or more growth promoting metabolites, which are essential to the growth of tree root mycorrhizae. It is also possible that some toxic compounds from fumigants were still present in the soil at inoculation time despite a four day aeration period. These toxic compounds were probably detrimental to the life and the development of the mycorrhizal fungi. gycorrhizal formation - The seedlings grown in pots inoculated with forest soil showed abundant mycorrhiza- tion of short roots at sampling time (Tables 16, 17). Very good mycorrhizal development was present on the roots of red pine seedlings grown in a pot with Rhizopggon roseolus inocu- lation. In pine these fungi formed compact mantles of mycelium on the surface of the short roots. The infected roots were shorter than the non-mycorrhizal roots and they were dichotomously branched once or many times. 83 Microscopic studies of mycorrhizae fixed in formalin - acetic acid - alcohol confirmed the visual observation. Those obtained following a Rhizopogon roseolus inoculation showed the hyphae mantle on the surface of the roots and also that the fungi had dissolved the middle lamellae of the epidermal and outer cortical cells and then the hyphae had surrounded the walls of those cells to form a pattern called a "Hartig net" (Figure 12). In non—mycorrhizal roots this pattern was not observed, but a regular arrangement of cells. At sampling time a large percentage of short roots of seedlings grown in fumigated soil inoculated with forest soil appeared to be ectotrophic mycorrhizae. But microsc0pic studies of these short roots did not show substantial evi- dence of true ectotrophic mycorrhizae. Hand-made cross- sections of these dichotomies showed a thin mycelium sheath and a disorganized cell structure. The cortex cells showed the presence of intra and intercellular hyphae, but most of the time in an irregular pattern (Figure 13). These hyphae could be due to a mycorrhizal fungus invading the cortex cells, but too young to have a definite pattern. These dicho— tomies could also be what are called: "ectendotrophic mycor- rhizae". The intracellular hyphae could also be hyphae from a pathogenic fungus. It is also probable that the use of a 84 .poou unoam may we mHHoU HMUHunoo ocsoum Auwc mHnuomV om£m>m “m .oommm umHsHHoomu#:H CHQH w¢ .AXoomv pom owumHsoooH mmmmmmmw mmmmmmmwmm :H meHmH mCHm own EOHM noon UHOSm ApfimHuV HoNHSHHOU>E pom amuonv HMNHfiuHooxelfloc mo mHOHuoomlmmOHU .NH ousmflm 85 Figure 13. Cross—sections of dichotomous short root of red pine showing (A) intra and (B) intercellular hyphae, and (G) a thin fungal sheath (3500K). 86 microtome would have given short root cross-sections of better quality and permitted an easier study of the phenomenon. Meanwhile we believe that these dichotomous short roots are true mycorrhizae, due to the morphological charac- teristics of these seedlings and the visual observation of mycorrhizal characteristics on the short roots. There is evidence that most of the time one cannot conclude to pres- ence of true ectotrophic mycorrhizae only by visual observa- tion, but the need of further microscopic studies is ob- vious. The red pine seedlings grown in pots inoculated with a pure culture of Amanita rubescens showed abnormal roots and malformed short roots which looked like mycorrhizae. These malformations could be due to incompatibility of this fungus with red pine or possibly it could also be related to the soil fumigation itself. Wilde and Persidsky (1956) reported mal- formations of mycorrhizae in Monterey pine seedlings following soil biocide treatment. A microscopic study of these roots showed that residual toxicants from the fumigant caused the formation of pseudo-mycorrhizae and malformations of the roots. The hyphae mantle and the Hartig net, characteristics of true ectotroPhic mycorrhizae, were absent. Meanwhile the cells seem normal inside these malformations (Figure 14). 87 AuHmHuw mcoHuuowlmwouo mnoHumeuomHme omofly «0 H H3 UoumHsoouH pom CH G30Hm mQHHooom omHm ooo mo .xxoommv .mmoomoflsu muHcoE< noon HoEHoch huonu .oH munon 88 Shoot phosphorus concentration in inoculated EQEE — Only the seedlings grown in pots kept as control and those where forest soil had been added at sowing time were analysed for P concentration. The data indicate that seed- lings grown in fumigated soil without any supplement have a very low P concentration and P uptake (Figure 15). This corroborates the P deficiency symptoms and the decreased growth observed at sampling time. The addition of 5.0 grams of forest soil per pot (2.0 kg) was related to a very good growth rate and a P concentration of 0.135 per cent for red pine and 0.165 per cent for white spruce. These results are generally in good agreement with earlier data. Ingestad (1962) reported that the optimum P range should be between 0.15 - 0.40 per cent in the pine leaves and 0.10 - 0.30 in the spruce leaves. Fowells and Krauss (1959) found 0.14 - 0.18 per cent as optimum in Pinus taeda and P. virginiana leaves. Leyton (1958) reported 0.15 per cent as optimum in Corsican pine leaves and 0.13 per cent as an optimum in Sitka spruce. Moderate deficiency corresponds to 0.08 - 0.15 per cent P and the minimum content seems to be about 0.05 - 0.06 per cent in pines (Ingestad, 1962). In spruce, Ingestad also reports that minimum content seems to be about 0.04 - 0.05 per cent and moderate deficiency corresponds to 0.07 - 0.10 89 PHOSPHORUS UPTAKE-MG/SHgOT O 23 8 § -L5D -I20 WWW (XMHROL RXESTSDK.AUNNON E] l L 2_J \MSPRUCE FUHNE g f I f ID 0 In 6 O N - _ 00.MJN£3M NOUNHUWEMKXDSNHOH£EWH R Figure 15. Influence of forest soil addition on the shoot P concentration and uptake of red pine and white spruce seedlings grown in vapam fumigated soil (Experiment 3). 90 per cent. In this study, at 0.04 per cent of P (control pots) the seedlings showed a severe deficiency and a poor growth (Figure 11). C- SUPERPHOSPHATE AND FOREST SOIL INOCULUM ADDITION TO VAPAM FUMIGATED NURSERY SOIL - GREENHOUSE (EXPERIMENT 4). This experiment was set to test the influence of different levels of forest soil inoculum and superphosphate, alone or in combination, on the growth and the development of red pine and white spruce seedlings grown in vapam fumi- gated soil. As in the growth chamber study the addition of forest soil inoculum increased the growth of both species of seedlings, but adding different levels of P resulted in a greater difference than soil inoculum in the development of the seedlings (Table 18). Soil inoculum addition (3.0 g per 2.0 kg soil/pot) significantly increased seedling height. However tripling the amount (9.0 g) did not bring a significant increase over the 6.0 9 addition (Appendix Table 29). When the dry shoot of both species is considered the soil inoculum was related to a significant increase (.05 level). For white spruce the low level (3.0 g) was also significantly different from the other two levels (6.0 and 9.0 g). Meanwhile the addition of soil inoculum improved the dry root weight of either red pine 91 oo mm« o.o mo on «.H HHo.o «o mo« o.o mo ke« o.H Hmo.o3 m.»mxse mHH vow N.¢H NoH mom o.o 0mm o.o om HNH mmm H.¢H mmH mom o.o mom o.o mH mOH mmm ¢.mH ooH mHn o.o omm o.m mH omH omm h.MH vow non ~.h mom I I 5H hHH hem m.mH va mmw m.n mom o.o oH mm hmv m.HH th lo m.n who o.o mH 00H nmo ¢.HH NmH mom m.h who o.m vH MHH mmv m.~H MhH mmo o.o who I I MH NOH owo h.HH mmH emu m.h mow o.o NH moH mom N.MH va mmh m.h moo o.o HH ONH woo m.mH NmH omo H.m wvv o.m 0H hNH mmm m.NH mmH moo o.o wow I I m om omv m.HH MHH 5mm «.5 wmm o.o m om omv n.HH omH moo N.n me 0.0 5 mm 5mm m.m mmH Hmm m.o VNN o.m 0 mm mom m.m mmH 0mm o.o wmm I I m 0v va o.m mm nmm m.o I I o.o v mo mmH H.@ mm mmv o.o I I o.o m mm mvH 0.5 mm nmm o.o I I o.m N mm mm m.m om VOH m.v I I I I HI me 08 EU mE me EU .. m£\mx . . m boom uoonm uanom “com pooam uanom m EszUooH Hflom oz unonz sue unons sun monumm ouHHB oCHm oom unweumoue .Hv uGoEHuomme HHOm oouomHEzm Emmm> m CH c3oum mmoHHooom monumm oUHHB ooo osHm own oHo mxoo3 mH mo mUHumHuouomumao HMUHmoHOHQHOE no mHo>oH mononmwonm ucoHoMMHo ooo EoHsooaH HHOm umouom mo uommmm .mH oHQmo 92 or white spruce seedlings, but not significantly. Moreover the different levels of soil inoculum are related to only a slight difference between them. There were significant differences (0.01 level) in morphological characteristics of the seedlings after P addi- tion. The height, dry shoot or dry root weight were much greater for seedlings of both species grown in soil with added P. A significant difference (.01 level) was found be- tween the low and the higher levels of P addition (Table 18, Appendix Table 29). A logarithmic equation (y - a + b log x) characterizes well the relation between the growth of seed- lings (shoot dry weight) and the P addition (Figure 16). Dark green and well balanced seedlings were obtained at the highest level with no evidence of a harmful effect (Figure 17). Benzian (1965), in England, reported that at the high rate of application (144 kg P/ha) Sitka spruce seed- lings grew exceptionally well. There was no indication of a harmful effect; on the contrary she thought that higher rates had to be applied to establish the full shape of the curve. At sampling time the presence of very few if any mycor- rhizae was observed on roots of seedlings grown in P fertilized soil. The addition of soil inoculum alone to the fumigated soil increased their numbers. This scarity of mycorrhizae can be related to the soil fumigation with vapam and also to the 93 .Hw ucoEHuomxmv HHOm ooummHEzm Emmm> m CH ooopm m mo ucsoem 0:» tom mmcHHooom museum ouH£3 pom mch own mo uanmB >uo uOOLm on» cooBaoQ coHumHom .oH ousmHm 29:84 «It oxomo «o.o o3. ¢«« 0 x P H P _ _ m o n N _ _ u _ NmJuNm X 00.4 8N+N® u F36 POOIm moamdm MEI; x wmquwu mzi ommIII x 004®mm+mm_u.._.>>n_ FOOIm IHVON r.OO¢ lfiuow .Ioom 9W ‘lHSIBM A30 IDOHS 94 . Awake ox ooo I 0 die 3 «so u m 4328 u < .EmHm SEE ox ooo u 0 Jim ox o«« I m .Houwoou u ¢ .uon “mHGoEHmouBV I HHom owsosnooum ooummHEzw Emmm> m CH dzoum mmGHHooom HuSmHuv ousumm ouHQB pom AuonV ocwm own so GOHuHoom m mo pummmm .hH wwzmHm 95 high P level in soil. Indeed mycorrhizae seem to need a low level of P to develop. Fowells and Krauss (1959) confirmed the findings of Hatch (1937) that mycorrhizae are generally more abundant with low levels of P and N. Hacskaylo and Snow (1959) further support these conclusions: prevalence of mycor- rhizae on pines was greatest in soils with no nutrient added. Phosphorus concentration in shoot tissue - The significance of different P level addition is shown in per- centage of shoot dry weight and in uptake in mg/shoot (Table 19, Appendix Table 30). Phosphorus addition to soil is related to a highly significant difference (.01 level) in shoot concentration and uptake for both red pine and white spruce. Low addition of P brought results significantly different (.01 level) from the highest level of P fertiliza- tion. Ingestad (1962) reported that a P concentration of .152 per cent corresponds to a moderate deficiency for pine. Tamm (1956) also reported that the deficiency level in cur- rent spruce leaves is 0.07 - 0.08 per cent. The values obtained were identical to those mentioned by these authors. The relation between soil P addition and shoot P concentra- tion of seedlings can be characterized by a logarithmic equation (Figure 18). The equation shows that these two variables are much more closely related for white spruce 96 Table 19. Effect of soil phosphorus addition on the shoot phosphorus concentration and uptake of red pine and white spruce grown in the greenhouse for 19 weeks; Experiment 4 (means of 4 replications). Treatment Shoot Phosphorusl % P Increase over No Phosphorus Conc.2’ Uptake Control kg/ha % mg/shoot Conc. Uptake RED PINE l 0 .152 .16 - — —. _ - - 2 224 .231 ** 1.22 ** 52 655 3 448 .234 ** 1.48 ** 54 820 4 672 .223 ** 1.46 ** 46 804 5 896 .245 ** 1.82 ** 61 1032 Tukey's (.05)' .042 .33 (.01) .055 .43 WHITE SPRUCE l 0 .069 .02 - - — — _ _ 2 224 .210 ** .64 * 204 2738 3 448 .246 ** 1.29 ** 256 5621 4 672 .266 ** 1.30 ** 285 5679 5 896 .305 ** 1.67 ** 342 7307 Tukey's (.05) .065 .53 (.01) .084 .69 l Determined on a dry weight basis. See Appendix Table 30 for statistical significance. 2 * Significantly greater than control at .05 level. ** Significantly greater than control at .01 level. 97 .Av ucoEHuomxmv HHOm ooummHesm Emmm> m 0» oooom m mo unseen on» one mmcHHooom oozumm ouH£3 com och ooh mo coHumuucoocoo m poonm oflu coo3uon coHumHom .mH ouson zeikfiq «IE oxooo «no 31. v«« o _ _ _ L _ x m c m N _ p p p p b x mow: roo.. xmxj_vmtw> .o «a . m .6& m m cm IH IOONW I. o a o a o X N x m oo....«e M x 004 .409; Icon. 0 83QO NE; on u” W 98 (R? = .90) than for red pine (R2 = .59). It seems that P concentration in white spruce was more influenced than in red pine by the level of P in soil. In a fumigated soil containing just a small amount of available P spruce grew very poorly and showed evident signs of P deficiency. The P uptake in seedling shoots in relation to the different levels of P addition gave significant differ— ences (.01 level) except for a few treatments (Appendix Table 30). The control seedlings had poor growth, low shoot P concentration, and consequently a very low uptake of P into shoots. Uptake at all the levels of P addition were different (.01 level) from the control. The in- crease in P taken up was from 655 to 1032 per cent depend- ing on the levels of P addition for red pine seedlings. For white spruce seedlings the percentage P increase was even much greater (2738 to 7307 per cent) after P addition to soil (Table 19). These facts give evidence of the highly beneficial effect of a high P addition to soil for the development of seedlings grown in a fumigated soil. D- SUPERPHOSPHATE ADDITIONS TO METHYL BROMIDE FUMIGATED SOIL - BERTHIERVILLE NURSERY (EXPERIMENT 5). This experiment was set to test the influence of different levels of P addition to nursery seedbeds, as 99 superphosphate, on the growth and development of red pine and white spruce seedlings grown in a methyl bromide fumi- gated soil. Due to very poor germination of white spruce results are shown only for red pine. Morphological characteristics - The addition of different levels of P increased significantly (.01 level) the growth and the development of seedlings. The height, the dry shoot and the dry root weight were much greater for red pine seedlings grown in soil with added P (Table 20, Appendix Table 31). There is even a signif- icant difference between low and higher levels of added P. The first two levels of P addition brought a sharp in- crease in the growth and the develOpment of the seedlings. The higher P additions were also related to an increase, but not at a proportional level. It appears that an op- timum level of growth has been attained after an addition of 672 kg P/ha and higher amounts have not added too much to seedling development. A logarithmic curve of the form y = a + b log x usually characterizes well this kind of response (Figure 19). At sampling time seedlings grown in plots with no added P had red purple needles, sign of a P deficiency. The plots where the low level of P (336 kg/ha) was added grew a 100 .Hw>oH Ho.o pm Houudoo smflu Houmoum ansmoHMHcmHm on .Ho>oH mo.o pm Houucoo cmfiu Houmoum >Huomowmocmfim e N .oUGMUHmHGmHm HMUHHmHumum Mom Hm oHQme xHocmmm< mom .mHme HHmHoB who o no oocwfiumquH om. Hmo. om mm m. AHo.V 3. «oo. 3 me n. «So. I, Pearce omHH 0mm 4% mm. 4* mmm. 4* Ho es mmH 4* m.v ommH o ova 00m 44 mo. *4 0mm. «4 mm 4* NnH 4* m.v ovMH m oHoH 00m 4* mm. 4* ohm. 4* n ye HmH 4* w.¢ mooH v oom mmm «4 mm. 4* Hmm. «e no «4 doH m.v who m «Hm on 4 mm. 4* mom. * mm moH N.v 0mm m I I I I oo. Hmo. m mm m.m o H NOO£m\fiE X m& WE EU M£\m¥ oxmumb .onou oxougn .ucou poem Hoofim mufimHom msuonmmonm 0% Hoeucoo um>o ommwhonH o N. Hmznofimm0£m HOOHm HHmHmZ Nun wcmsumwum. 9.3 .AmcoHuooHHmoH o mo mnmoEV sH c30um mmnHHooom omHm ooh mo .moHumHumuomum£o HMUHmoHOHQHOE m «soEHuomxm amxwo3 hH now momflommm hummuoa oxnumz ocm noHumHucoocoo monoxmmosm uoogm ox“ CO QOHHHUUm monogmmOHQ HHOm mo Hommmm .ON THQMH .Hm pcoEHuomxmv HHOm Hummus: poummHEsm ooHEOHH HxnuoE m on oooom m m0 uczoem oflu ooo mmcHHooom mch own oHo mxmoz nH mo uzmHo3 Hap uoonm onu cooBqu coHumHom .mH ousmHm 101 205.504 41?. cxomm. .¢Vn_ moo. th wmm O H H H H H H x w m v m N H H H H H H H Ton mmdwm r g DW'JJ'OBM A80 1.0045 me%_mmrmmflr I 8 ICON 102 few red purple seedlings, but the majority of them was green. The plots with higher rates of P grew dark green and well developed red pine seedlings. In plots kept as control we got seedlings of small size (dry weight) based on the stand- ards set by Armson and Carman (1961). On the contrary the seedlings, grown in plots having received 672 kg P/ha or more, were of very large size when compared to the same values. No mycorrhizae were present on the short roots at sampling time. This is probably due to the fumigant effect on mycorrhizal fungi and also for a few treatments to the high level of P in the soil solution. Shootgphosphorus concentration - Analysis of shoots confirmed a P deficiency condition in the seedlings grown in plots that were not fertilized with P (.091 per cent P concentration). Ingestad (1962) gave a level of P of 0.08 to 0.15 per cent as an evidence of deficiency in current leaves of pines. In this experiment the addition of the lowest level of P brought a major increase in the P concentration of pine leaves (.243 per cent). The higher P additions still increased the level of P in the pine needles (Table 20). These later values are in the range for an op- timum growth (Armson and Carman, 1961; Ingestad, 1962). 103 As mentioned the addition of P brought a significant difference (.01 level) on the concentration of P in the pine leaves (Appendix Table 31). The lowest level of P addition (336 kg/ha) is related to an increase of 167 per cent in the P concentration in the seedling shoots. This increase goes to 320 per cent with the highest P addition to soil. Greater differences are shown for the P uptake in the seedling shoots (Table 20). The relation between the soil P addition and the shoot P concentration and uptake is best characterized by a logarithmic curve (y = a + b log x). Indeed we got R2 values of .87 and .86 respectively (Figure 20). A sharp increase is related to the first P additions, and a much lower re- sponse to the last ones. This finding suggested an examina- tion of the relation between the shoot P concentration (% D. WT.) and the shoot weight. Phosphorus concentrations of the red pine seedlings showed a consistent trend of in- creasing concentration with increase in seedling size; a trend characterized by a linear equation (Figure 21). Armson (1968) found a similar trend for white spruce, whereas for red pine the concentrations decreased somewhat. This relation seems to be reasonable in light of the im- portance of P for the plant. Phosphorus as adenosine 104 .Hm ucoEHuomxmv HHOm Hummus: poummHE3m prEOHQ Hxnuoe m on oooom m mo unsoem ozu pom mmcHHpoom 203.50%ch ooo mo oxmumz m ooHu Hocm coHumuucoocoo m Hooch ocHu cooBqu coHuoHom .om ouomHm <1». oxowo. eon. moo. «Ho omm r H H H H H x o o v n N H _ H _ _ owl H.00.. oorum o N x 001. .98." 9.535 a 0 ¢. 1 km." a N x 00.. Ot+m=.n§.v .0200 a PHOSPHORUS UPTAKE-MG/SHOOT 8, I 0Q] . ICON .room. 109v. (%) .LOOHS NI NOILVBLNBDNOO SDUDHdSOHd loom. 105 .Hm acoEHuomme mGCHHooom mch ooh oHo mxoo3 5H mo uanoz uoonm on» new uoonm CH coHumuucoucoo m may coo3uon coHumHom .HN ousmHm oz skim—m3 .HbOIm OON oo. 00. On H H H H No.0 "NC x NOO.+O_O. a» ‘J. M '0 m SDUOHdSOHd 106 triphosphate and numerous phosphorylated products partici- pate in nearly all synthetic reactions of the plant cell. It is essential for the development of the meristematic tissues and it is associated with the general process of respiration. It is apparent that adequate P nutrition will be related to a better development and growth of conifer seedlings. CHAPTER.V CONCLUSIONS AND IMPLICATIONS FOR MANAGEMENT The principal findings of this study are: - Soil sterilization (methyl bromide, vapam or heat) severely depressed the development of red pine and white spruce seedlings grown in nursery soil without supplemen- tal P. — The addition of N alone to sterilized soil did not improve the growth of either red pine or white spruce. In combination with the P sources N generally appeared to en- hance the development of the seedlings and their uptake of N. Ammonium-N was a superior source of N to N03-N. - In fumigated soil red pine and white spruce seedlings raised with no supplemental P were P deficient according to shoot analysis. - The combination of soil sterilization, NH4-N and superphosphate fertilization produced larger seedlings and higher shoot P contents than any other treatment. 107 108 — Superphosphate was clearly the best source of P. The relatively cheap rock phosphate was associated with a better seedling develOpment and P uptake than bone meal. - Inoculation of fumigated soil with a small quantity of forest soil was associated with dark green seedlings showing a much better growth and higher P uptake than those from con- trol or pure culture inoculation treatments. Nursery Culture Implications Soil fumigation is widely used in forest nurseries to control nematodes, damping—off, root rot fungi, and weeds. Occasionally fumigation is followed by strongly adverse re- sponses. Entire beds of seedlings fail to grow much beyond the cotyledon stage, become purplish in color, and commonly die over winter or grow very poorly the next season. Phosphorus is one of the most important elements in plant nutrition. All the experiments showed that the uptake of this element tends to be hindered by fumigation of forest nursery soil while it is normal in the untreated soil. This low P uptake is reflected in poor seedling growth, visual P deficiency symptoms, and low shoot P concentration. In all the experiments it appeared that white spruce seedlings were more influenced than red pine by nursery soil 109 fumigation and low level of P; white spruce was also more responsive to high P addition. It seems that the addition of a very small quantity of forest soil might be an efficient way of supplying mycor- rhizal fungi inoculum to nursery soil. The use of soil from forest stands may introduce species of symbiotic fungi that would be well adapted to the environmental conditions en- countered following seedling out-planting. This inoculation with forest soil would also strongly influence the microbial recolonization of nursery soil. This inoculation method can also present certain disadvantages. The cost of obtaining soil from forest stands and its application to nursery seed- beds can be high. Furthermore there also is the danger of reintroducing pathogens into seedbeds. Pathogens could be- come more virulent in the artificial nursery environment than in the natural forest soil. A more effective way to inoculate fumigated nursery soil with pure cultures of mycorrhizal fungi is needed. This implies isolation of competitive mycorrhizal fungi well adapted to the species being grown. A pure culture inocula- tion technique adapted to practice would require the develOp- ment of a growing culture medium that could be easily trans- ferred into the nursery soil. 110 These studies showed that symptoms of P deficiency of seedlings can be overcome in part by an application of rock phosphate or bone meal, but only superphosphate fer- tilization significantly increased the seedling biomass and the shoot P concentration. At the same time NH4-N seemed to be a better N source than NO3-N. In nursery experiments the seedling biomass and the shoot P concentration of conifer seedlings were closely re- lated to the amount of P (superphosphate) added to fumigated nursery soil. The optimum level of P was 672 kg P/ha for conifer seedlings, although healthy seedlings and adequate shoot P concentration were obtained with lower rates. A good way to get this high rate of P into the soil is to drill superphosphate alongside the seeds, at sowing time. We found that it was difficult with mechanical seeders to band the P fertilizer close enough to the seeds. A suggestion not tried in these experiments would be to coat the seeds with finely ground superphosphate. The experiments show that shoot P concentration is correlated with red pine seedling weight. This is another indication of the necessity of adequate P fertilization for optimum conifer seedling growth. Furthermore suitable P 111 fertilization and seedling P uptake may significantly influence: (1) seedling survival, (2) resistance to disease, and (3) re- sistance to insect attack. In addition an adequate amount of available P in fumigated nursery soil may influence a delayed nutritional gain in subsequent growing seasons in the nurs- ery and even after outplanting in the field. This will shorten the period needed to raise seedlings in the nursery and permit the production of more uniform stock. This research may provide useful guidelines to nurs- erymen who encounter stunted and P deficient seedlings follow- ing soil fumigation. It may also provide some guidelines for investigators of mycorrhizal fungi. LITERATURE CITED LITERATURE CITED Alexander, Martin. 1959. Herbicides and soil microorganisms. Farm Research XXIV: 15. Armson, K.A., and R.D. Carman. 1961. Forest tree nursery soil management. Ontario Department of Lands and Forests, Toronto. Armson, K.A. 1968. 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Effect of fumigation with trizone on micrdbial properties of soil and growth of seedlings. Down to Earth 20: 13-15. Zak, B. 1964. Role of mycorrhizae in root disease. Ann. Rev. Phytopathol. 2: 377-392. APPENDIX 119 Appendix Table 21. Preparation of "Hagem" agar for culture of mycorrhizal fungi. “v Ina—H...- Agar 15.0 gm Glucose 5.0 gm Malt Extract 5.0 gm P ." KH2 O4 0 3 gm .7 .5 MgSO4 H20 0 gm NH Cl 0.5 gm 4 FeCl3 (1% solution) 0.5 ml H O to 1000 ml 2 120 Homzm HUGH m2 mm mfi mz m2 m2 H m m> m we mz m; a. 4. oz H m .muo m> m .muonH «e to ya on *4 to H COHHHUOM m m> m OZ lm. NZ 0? Roman Uou oz 4 oz mz oz oz H m m> x m oz oz oz oz * 44 H m .muo m> m .ouocH to s so so to s* H GOHHHGUM m m> m OZ NZ HoucoEonmom HDOHHHZ «e st * mZ so *e H mOZ m> #32 to to es to es s* H Z m> Z HDO\3 so so so so es es N Nm. 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